A simple and sensitive sensor has been successfully developed for fast monitoring of azathioprine (AZA) by combining valuable properties of electrospun poly (vinyl alcohol)/chitosan (PVA/CS) nanofibers and surface plasmon resonance of silver nanoparticles (AgNPs) as a smart probe. Attachment of silver ions onto the surface of PVA/CS nanofibers via anchoring with free amino groups in the chitosan framework, and then reducing by ascorbic acid resulting in the formation of decorated AgNPs onto the surface of nanofibers. The presence of AZA during reduction process leads to a decrease in the formation of AgNPs onto the surface, consequently decreasing plasmon resonance intensity of nanoparticles. This change is utilized as a means to create an environmental friendly hybrid probe for the fast and careful detection of AZA. Developed plasmonic nanofibers exhibit a linear response for AZA concentration in the range of 0.14-2.88 mu M with the detection limit of 0.09 mu M. The relative standard deviation corresponding to eight replicate determinations of 0.86 mu M and 2.88 mu M of AZA was 1.93 and 2.70%, respectively. Suggested method was effectively applied for the accurate determination of AZA in tablet formulations and human serum samples.