Abstract: The pH value of cow manure compost products is relatively high, and the traditional aerobic composting process tends to lead to nutrient loss, limiting the improvement of organic fertilizer quality. The addition of acid-producing bacteria can reduce the pH value and nitrogen loss in cow manure compost to some extent, but its regulatory effect is limited in traditional composting processes. This study isolates and screens lactic acid bacteria from fresh cow manure, exploring the effect of a composite lactic acid bacteria agent combined with an anaerobic fermentation pretreatment process on cow manure composting. Four treatments were designed: T1, the material was added with a composite bacterial agent and underwent anaerobic fermentation for 4 days before aerobic fermentation; T2, the composite bacterial agent was added followed by aerobic fermentation with ventilation; T3, sulfuric acid was added followed by aerobic fermentation with ventilation; T4, sulfuric acid and the composite bacterial agent were added and mixed thoroughly before aerobic composting. The original material aerobic composting was used as a control (CK). The results show that 21 suspected lactic acid bacteria strains were isolated from fresh cow manure. Two target strains were obtained through initial and secondary screenings, based on rapid growth and strong acid production ability. These strains were identified as Lactobacillus amylovorus LB31 and Weissella paramesenteroides BY1, with the optimal viable cell ratio of the composite bacterial agent prepared from the two strains being 7:3. The pH value of the material after anaerobic fermentation pretreatment decreased from 7.67 to 7.38, with T1 showing a 4.07% reduction compared to CK. At the end of composting, compared to CK, the T1 treatment showed the least carbon and nitrogen loss, which decreased by 27.53% and 37.50%, respectively. The mass fractions of available phosphorus and available potassium increased by 14.68% and 10.95%, respectively, and the pH value decreased by 0.46. In conclusion, adding a 3.0% mass fraction of composite lactic acid bacteria agent to cow manure compost, followed by 4 days of anaerobic fermentation and aerobic composting, can significantly reduce the pH value of the compost material and product, and decrease nutrient loss during composting. This study provides bacterial strain resources for the application of lactic acid bacteria in composting, while also offering theoretical and technical foundations for combining anaerobic and aerobic processes to reduce compost pH and nutrient loss.