A 530 nm light emitting diode was coupled to a microfluidic sensor to facilitate photolysis of nitrosothiols (i.e., S-nitrosoglutathione, S-nitrosocysteine, and S-nitrosoalbumin) and amperometric detection of the resulting nitric oxide (NO). This configuration allowed for maximum sensitivity and versatility, while limiting potential interference from nitrate decomposition caused by ultraviolet light. Compared to similar measurements of total S-nitrosothiol content in bulk solution, use of the microfluidic platform permitted significantly enhanced analytical performance in both phosphate-buffered saline and plasma (6–20× improvement in sensitivity depending on nitrosothiol type). Additionally, the ability to reduce sample volumes from milliliters to microliters provides increased clinical utility. To demonstrate its potential for biological analysis, this device was used to measure basal nitrosothiol levels from the vasculature of a healthy porcine model.