An indirect high-performance capillary electrophoresis with amperometric detection (CE-AD) method has been developed for determination of lactic acid (LA) in body fluids of lactating postpartum women. Several important factors such as the running buffer additive and concentration, the working electrode potential, the pH value and concentration of the running buffer, the separation voltage and injection time were investigated. Under the optimum conditions, LA could be well separated with co-existing interferences including uric acid (UA) in real samples in a 90-cm-length capillary at separation voltage of 12 kV in 4.0 × 10−6 g mL−1 3,4-dihydroxybenzylamine (DHBA)/40 mmol L−1 H3BO3–Na2B4O7 buffer (pH 7.8). The linearity between peak current and concentration of LA was over three orders of magnitude with detection limit of 5.00 × 10−7 g mL−1 (S/N = 3). This proposed method has been successfully used to study the effects of moderate exercise on LA content in breast milk and urine samples of lactating postpartum women, and assay results showed that LA content in breast milk can return to normal level through 60 min rest without decreasing acceptance by breast-feeding infants, although the LA level did increase by 4–6 times in both breast milk and urine samples at 10 min after moderate exercise.