This study focused on the development and validation of a nutrition literacy assessment instrument for Chinese lactating women (NLAI-L). A comprehensive literature review and group discussion by experts in relevant fields were adopted to determine the dimension, topics and questions of NLAI-L. Content validity was evaluated by a panel of experts. The exploratory factor analyses (EFA) and confirmatory factor analyses (CFA) were used to evaluate the construct validity. Cronbach’s α and split-half reliability were applied to examine the reliability of NLAI-L. The final NLAI-L consisted of 38 questions covering three dimensions: knowledge, behavior and skill. The EFA revealed four sub-domains for knowledge, one sub-domain for behavior and four sub-domains for skill. The results showed that NLAI-L had satisfactory content validity (CVI = 0.98, CVR = 0.96), good reliability (Cronbach’s α coefficient = 0.84) and acceptable construct validity (χ²/df = 2.28, GFI = 2.81, AGFI = 0.79, RMSEA = 0.057). In the application part, the average NL score was 46.0 ± 9.3. In multivariate linear regression, education level, age, postnatal period and occupation were the potential influencing factors of NL for Chinese lactating women. The study established an effective and reliable assessment instrument for Chinese lactating women (NLAI-L) through qualitative and quantitative methods. The establishment of NLAI-L will provide an effective tool for exploring the role of NL in health or disease and provide a basis for the formulation of targeted nutrition interventions.