Abstract: Plant-derived peptides have been the high-quality resources of plant protein, because of their natural availability, unique nutritional and physiological functions, particularly without any toxic or side effects. The high protein content can also be balanced in the composition of amino acids. Among them, rapeseed/canola meal is one of the by-products in the extraction of rapeseed oil, while commonly used as animal feed, resulting in low economic benefit. Rapeseed protein hydrolysates have been proven to process antioxidant activity. High-value use of rapeseed protein source is very necessary. Enzymatic hydrolysis can be preferred to produce the bioactive peptides, due to the high specificity, mild conditions, and no by-products. However, the preparation of rapeseed active peptides usually requires the rapeseed protein prior to enzymatic hydrolysis, which is relatively complicated and consumed. Our previous study found that the rapeseed meals with superfine grinding effectively increased their protein dissolution rate. In this study, more attempts were made to directly prepare the active peptides by enzymatic approaches. The raw material was taken as the rapeseed meal powder that was treated with superfine grinding. A comparison was made on the molecular weight distribution and antioxidant activity of the products after rapeseed meal enzymatic hydrolysis by different enzymes. Furthermore, the high-purity antioxidant peptides were purified from the enzymatically hydrolyzed products by membrane separation and Sephadex G-15 column chromatography. The antioxidant activity was evaluated by DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging capacity, ABTS (2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging capacity, and total antioxidant activity (T-AOC). Finally, the amino acid sequence of the peptide was determined by LC-MS/MS. The results showed that the proteolytic products of rapeseed/canola meal with the higher peptide content were obtained by direct proteolytic hydrolysis of the rapeseed/canola mean ultrafine powder using alkaline protease and flavourzyme. Both hydrolytic products showed better ABTS free radical scavenging and total antioxidant at 1 mg/mL, but the lower DPPH free radical scavenging. Moreover, the hydrolytic products of flavourzyme were mainly composed of small peptides with a molecular weight of less than 1 kDa. The alkaline protease hydrolysis was dominated by peptides from 5 to 10 kDa fractions. The antioxidant activities of peptides were related to their molecular weight distribution. Specifically, the low molecular weight fraction of alkaline protease hydrolysis (＜1 kDa) showed better ABTS free radical scavenging at (89.73±0.33)% and total antioxidant activity at (2 669.5±2.82) μmol/g at 1 mg/mL. Furthermore, the active peptide fraction (J3) was achieved using Sephadex G-15 gel filtration chromatography. The peptide content of the J3 fraction was about 32.5%. Five peptides were selected, namely RF, LPF, PAGPF, VF, and LATPF. All five peptides showed a content of over 50% hydrophobic amino acids with phenylalanine as the N-terminal. Specially, RAGPF, VF, and LATPF were newly identified. In summary, the rapeseed meal can be used as a source of prepared antioxidant peptides, which would broaden their applicability. This finding can provide a theoretical reference to further develop the rapeseed/canola meal protein resources through direct enzymatic hydrolysis of rapeseed meal ultra-fine powder.