The rapid development of both wearable and implantable biofuel cells has triggered more and more attention on the lactate biofuel cell. The novel lactate/oxygen biofuel cell (L/O-BFC) with the direct electron transfer (DET)-type lactate oxidase (LOx) anode and the platinum group metal (PGM)-free Fe-N-C cathode is designed and constructed in this paper. In such a reasonable design, the surface-controlled direct two-electron electrochemical reaction of the lactate oxidase was determined by cyclic voltammetry (CV) on the carbon nanotube (CNT) modified electrode with favorable high electrochemical active surface area and electronic conductivity. Additionally, the biosensor based on DET-type LOx modified electrode impressively presented linear response to lactate with different concentrations from 0.000 mM to 12.300 mM. In particular, the apparent Michealis-constant (KMapp) calculated as 0.140 mM clearly indicates that LOx on CNT has strong affinity to the substrate lactate. Meanwhile, 4e- transfer oxygen reduction reaction (ORR) was proven to take place on the Fe-N-C catalysts inthe 0.1 M PBS system, indicating the advantage by using the Fe-N-C catalysts at the cathode of L/O-BFC. Last but not least, the L/O-BFC with the direct electron transfer (DET)-type lactate oxidase(LOx) anode and the Fe-N-C cathode produced an superior open circuit potential (OCP) of 0.264 V and a maximum output power density (OPD) of 24.430 µW cm-2 in O2 saturated 95.020 mM lactate solution. The above results will not only bring about significant interest in developing a DET-type biofuel cell, but also offer guiding direction to explore novel catalyst materials for the biofuel cell. This work enriches the research content and may push developments of the implantable and wearable biofuel cell forward.