Nanofluidic hybrid membranes display distinct ionic current rectification (ICR) properties and provide high surface area for immobilizing probes on the outer surface, exhibiting great potential in detection of biomolecules. Herein, we fabricated MOFs/AAO hybrid membrane with aptamers functionalized on the outer surface for in situ detection of living cells released secretions. TNF-α (a small molecular protein secreted by macrophages) was used as a model. After TNF-α was specifically captured by aptamers on the membrane surface, the asymmetry of surface charge on the hybrid membrane was amplified, the ICR was increased from 3.89 to 18.85. According to the ICR change, TNF-α was sensitively measured with a detection limit of ∼0.49 pM, which was significantly lower than other reported methods. When the hybrid membrane was clamped in the middle of self-made device, PET membrane incubated macrophages was rolled up and inserted into the chamber to mimic cellular microenvironment. Macrophages released TNF-α could be real time monitored with ionic current, macrophages and normal cells could be effectively distinguished according to the released TNF-α level. Thus, we proposed a nanofluidic platform for accurately measuring cell secretions in an engineered cellular microenvironment with a direct manner, without the need for labels or amplification steps.