Reliable and sensitive monitoring of organophosphorus pesticide (OP) residues in tea is essential for food safety and public health protection. Despite their effectiveness, the performance of these electrochemical sensors based on acetylcholinesterase (AChE) is often limited by insufficient interfacial enrichment and slow charge transfer kinetics. In this study, hollow spherical nitrogen-doped porous carbon (HS-NC) derived from ZIF-8 was employed as an interfacial matrix for parathion-methyl (PM) detection. The hollow porous structure favours the immobilization of enzyme and molecular diffusion. The nitrogen speciation controls the interfacial adsorption and electron transfer. By combining experimental and theoretical analysis, distinct functionalities of different nitrogen configurations are revealed: pyridinic-N plays a dominant role in PM adsorption and interfacial pre-concentration, while graphitic-N contributes to charge transport in the conjugated carbon network. By benefiting from the synergistic interfacial regulation, the AChE-CS/HS-NC/GCE achieves a broad linear range and a low detection limit of 1.77 × 10-15 M toward PM. Furthermore, the biosensor shows good reproducibility, stability, selectivity, and reliability for real tea samples. This study presents a nitrogen-speciation-guided interface-engineering protocol to improve enzymatic electrochemical sensing, paving the way for new structure–function insights in heteroatom-doped carbon interfaces.