A lactate self-powered biosensor was assembled for the detection of lactate in sweat based on flexible textile matrix enzymatic electrodes. A porous three-dimensional electrode with high flexibility and electrical conductivity was obtained by decorating composites of reduced graphene oxide and carboxylate multi-walled carbon nanotubes onto a cellulose fabric substrate. The excellent enzyme embedding method using gel electrolyte showed a Michaelis-Menten constant of 1.46 mM, which implies that lactate dehydrogenase had a high enzymatic activity. The assembled self-powered lactate biosensor showed a sensitivity of 3.16 µW mM^− 1 cm^− 2 in the 0–10 mM linear range and a detection limit of 9.49 µM (S/N = 3). Meanwhile, the biosensor has good tensile flexural stability, selectivity, and long-term stability. The lactate self-powered biosensor recovered 99.66–108.43% of lactate from real sweat with a relative standard deviation of less than 4.65% when integrated into clothing. The biosensor is promising for sensing lactate in real sweat.