Abstract: Strawberry (Fragaria × ananassa Duch.) is one of the most valuable fruits with unique tastes and high nutrient contents. The strawberry rich in anthocyanins can also be used to prevent diabetes, cancer, inflammation, and nervous system diseases. Traditionally, organic solvents are utilized to extract anthocyanins, such as methanol, ethanol and acetone, leading to environmental and health issues, as well as the high costs of solvent removal. Alternatively, the green solvent glycerol has been commonly used in food, cosmetic, and pharmaceutical fields, due to its biodegradability, non-toxicity, and low cost. Furthermore, an acidic environment can also provide for the preservation of anthocyanins, where glycerol was acidified by citric acid. In the present study, a one-step protocol was proposed to extract and preserve the anthocyanins from ‘Benihoppe’ strawberry, according to the four individual factors, such as concentration of glycerol, extraction temperature, liquid-to-solid ratio, and ultrasonic time. Single-factor experiments were first conducted to optimize the extraction, according to the yield of anthocyanins. The response surface method (RSM) was then carried out for further optimization. The anthocyanins were identified from the acidified glycerol extracts using the UPLC-Triple-TOF/MS equipment. A systematic investigation was made to explore the effects of acidified glycerol on the stability of anthocyanins under different temperatures, pH, and light conditions. Moreover, molecular dynamics and quantum chemical calculations were used to clarify the intrinsic mechanism of the extraction and preservation of strawberry anthocyanins by acidified glycerol. The results indicated that the four influencing factors on the yield of anthocyanins were ranked in the descending order of the extraction temperature > ultrasonic time > concentration of glycerol > liquid-to-solid ratio. The yield of anthocyanins was dominated by the individual factors (concentration of glycerol, extraction temperature, and ultrasonic time), interactive factors (concentration of glycerol and extraction temperature), and the quadratic factors (concentration of glycerol, and extraction temperature), according to the ANOVA. RSM results showed the optimal factors were achieved as follows: concentration of glycerol was 26%, extraction temperature was 67 °C, liquid-to-solid ratio was 34 mL/g, and ultrasonic time was 53 min. At the same time, the predicted and actual maximum yield of anthocyanins were (895.38±1.56), and (902.41±0.84) μg/g, respectively. Four anthocyanins were identified by the UPLC-Triple-TOF/MS, including delphinidin-3-glucoside, cyanidin-3-glucoside, pelargonidin-3-glucoside, and malvidin-3-glucoside. The Ultra Performance Liquid Chromatography (UPLC) was also used to quantify the delphinidin-3-glucoside (57.45 μ/g), cyanidin-3-glucoside (125.38 μg/g), pelargonidin-3-glucoside (619.53 μg/g), and malvidin-3-glucoside (96.12 μg/g). Stability tests demonstrated that the acidified glycerol significantly improved the storage stability of anthocyanins under light conditions at high temperatures, compared with the methanol solvent. The molecular dynamics showed the glycerol system shared the larger diffusion coefficient of 0.57 m²/s, a hydrogen bond number of 406, and a smaller intermolecular interaction energy of -1798.85 kcal/mol. In summary, the green solvent of acidified glycerol can be expected to more efficiently extract and preserve anthocyanins from strawberries in industrial applications.