Multi-walled carbon nanotubes (MWCNT) play a synergistic role with conducting polymer in practical applications such as biological sensing. In this paper, multi-walled carbon nanotube and polypyrrole (PPy) composites were prepared on a fiber surface for the first time, and their morphology and electrical properties were characterized. Compared with PPy-coated fiber, the presence of carbon nanotubes induced the growth of large areas of PPy nanowires. In addition, fiber organic electrochemical transistors (FECTs) based on PPy and MWCNT were assembled, showing a higher on/off ratio, better stability, and greater flexibility. The lactate biosensor based on FECTs exhibits high sensitivity, with a correlation coefficient of R = 0.9889, quick response time of 0.6-0.8 s, a wide linear response range of 1 nM-1 mM, and excellent selectivity for lactate. Furthermore, the lactate concentration in human sweat was successfully detected by a FECT-based sensor. The hybrid fibers can be easily woven and placed on fabric simply by stitching. This favorable performance of the FECT-based sensor makes it suitable for noninvasive sensing of lactate. Therefore, it provides a promising platform for future use in healthcare and detection applications. Graphical abstract.