Real-time monitoring of nitric oxide (NO) is of great importance in diagnosing the physiological functions of neurotransmission, cardiovascular, and immune systems. This study reports the carbon nanotube-interconnected ruthenium phthalocyanine nanoparticle nanocomposite and its applicability in construction of an electrochemical platform, which could real-time detect NO released from the vascular endothelial barrier (VEB) model in cell culture medium. The nanocomposite exhibits regular morphology, uniform particle size, and excellent electro-catalytic activity to electrochemical oxidation of NO. Under optimal conditions, the electrochemical device has high sensitivity (0.871 μA μM−1) and can selectively detect NO down to the concentration of 6 × 10−10 M. The human brain microvascular endothelial cells were cultured onto the Transwell support to construct the VEB model. Upon stimulated by L-arginine, NO produced by the VEB diffuses into the bottom chamber of the Transwell, and is real-time monitored by the electrochemical device. Moreover, evaluation of the NO inhibition by drug is realized using the electrochemical device-Transwell platform. This simple and sensitive platform would be of great interesting for evaluating the endothelial function or its pathological states, and screening the related drugs or chemicals.