By enhancing the speed, accuracy, and sensitivity, it opens up a new possibility for direct analysis of biologically relevant entities such as nucleic acids and proteins in single cell level. Nanofluidic systems with critical dimensions which were comparable to the molecular scales can provide the aforementioned performance, offering a novel basis for ultrasensitive and high-resolution bio-detections and medical diagnostics. Inspired by this concept, a nanofluidic system, integrated with a novel multi-nanochannel filter, an electrokinetic nano-preconcentrator, and a nanofluidic DAEKF detector for the detection of single cell releasing, is developed. A diluted solution of culture medium from PC-12 cells excited with excess nicotine concentration was detected with near single cell level amounts (c.a. 3000-10000 molecules) in this system after all processing in 20 min. Results showed that this multi-depth micro/nanofluidic chip exhibits superior sensitivity, efficiency, reliability, and reproducibility than those of conventional microchip electrophoresis /electrochromatography systems.