This study aims to explore the feasibility and accuracy of utilizing large language models (LLMs) to assess the risk of bias (ROB) in cohort studies. We conducted a pilot and feasibility study in 30 cohort studies randomly selected from reference lists of published Cochrane reviews. We developed a structured prompt to guide the ChatGPT-4o, Moonshot-v1-128k, and DeepSeek-V3 to assess the ROB of each cohort twice. We used the ROB results assessed by three evidence-based medicine experts as the gold standard, and then we evaluated the accuracy of LLMs by calculating the correct assessment rate, sensitivity, specificity, and F1 scores for overall and item-specific levels. The consistency of the overall and item-specific assessment results was evaluated using Cohen’s kappa (κ) and prevalence-adjusted bias-adjusted kappa. Efficiency was estimated by the mean assessment time required. This study assessed three LLMs (ChatGPT-4o, Moonshot-v1-128k, and DeepSeek-V3) and revealed distinct performance across eight assessment items. Overall accuracy was comparable (80.8%–83.3%). Moonshot-v1-128k showed superior sensitivity in population selection (0.92 versus ChatGPT-4o’s 0.55, P < 0.001). In terms of F1 scores, Moonshot-v1-128k led in population selection (F = 0.80 versus ChatGPT-4o’s 0.67, P = 0.004). ChatGPT-4o demonstrated the highest consistency (mean κ = 96.5%), with perfect agreement (100%) in outcome confidence. ChatGPT-4o was 97.3% faster per article (32.8 seconds versus 20 minutes manually) and outperformed Moonshot-v1-128k and DeepSeek-V3 by 47–50% in processing speed. The efficient and accurate assessment of ROB in cohort studies by ChatGPT-4o, Moonshot-v1-128k, and DeepSeek-V3 highlights the potential of LLMs to enhance the systematic review process.