Selective and sensitive determination of L-cysteine (L-Cys) plays an important role in clinical diagnoses and regulation of L-Cys level. However, glutathione (GSH) interferes greatly with the L-Cys detection due to their similar electrochemically active functional sulfhydryl groups and the high abundance of GSH in biological samples. Herein, Ni2P nanocrystallines anchored on black phosphorus nanosheets (Ni2P @ BPNSs) electrocatalysts were prepared by an in-situ solvothermal method using BPNSs as phosphorus sources and substrate. The electrochemical behaviors of Ni2P @ BPNSs with different Ni2P contents were studied for L-Cys determination. The experimental and DFT calculation results reveal the extraordinary electrocatalytic and selective sensing mechanism of Ni2P @ BPNSs toward L-Cys is attributed to the high selective adsorption and electrocatalytic activity of Ni active sites, and BPNSs matrix. The electrochemical sensor displays low detection limit (2 nM), more negative oxidation potential (+0.30 V), extremely broad linear range (0.02 −1000 μM), excellent sensitivity (396.00 μA mM−1 cm−2), and extraordinary selectivity. The sensor has been practically applied in detection of L-Cys concentrations in serum and urine samples. This work opens the avenues for the engineering design of metal atoms decorated BP materials in biosensors with enhanced sensing performance by adjusting the properties of materials, allowing for more selective and sensitive analyte detection.