In this work, we developed a novel and facile strategy for the synthesis of a highly active and stable electrocatalyst based on PdCu alloy nanoparticles (PdCu-ANPs) embedded in 3D nitrogen-doped carbon (NC) nanofoam arrays (NFAs), which were assembled on flexible carbon fiber (CF) microelectrode for in situ sensitive electrochemical detection of biomarker HO in cancer cells. Our results showed that NC-NFAs support possessed a unique hierarchically porous architecture by integrating the macrospores in arrays scaffold within mesopores in individual NC nanofoam, which offered exceptionally large surface area for embedding high-density PdCu-ANPs in it as well as facilitated the mass transfer and molecular diffusion during the electrochemical reaction. Taking the advantages of the unique structural merit of NC-NFAs support and excellent electrocatalyitc properties of PdCu-ANPs that embedded in it, the resultant PdCu-ANPs/NC-NFAs modified CF microelectrode exhibited good electrochemical sensing performances towards HO including a wide linear range from 2.0 μM to 3.44 mM, a low detection limit of 500 nM, as well as good reproducibility, stability and anti-interference ability. When used in real-time in situ tracking HO secreted from different types of human colorectal cancer cells, i.e., HCT116, HT29, SW48 and LoVo, it can distinguish the types of cancer cells by measuring the number of extracellular HO molecules released per cell, which demonstrates its great promise in cancer diagnose and management.