In this study, the patulin imprinted and the non-imprinted nanoparticles are synthesized by the two-phase mini emulsion polymerization method and characterized by zeta-size analysis, Fourier transform infrared spectroscopy, and scanning electron microscopy. Afterwards, the patulin imprinted and the non-imprinted nanoparticles are attached on the surface of surface plasmon resonance (SPR) chips. The patulin imprinted and the non-imprinted SPR nanosensors are characterized by using atomic force microscope, ellipsometer, and contact angle measurements. Kinetic studies for patulin detection are carried out in the concentration range of 0.5 nmolar-750 nmolar. The limit of detection and the limit of quantification values are obtained as 0.011 nmolar and 0.036 nmolar, respectively. In all kinetic analysis, the response time is 13 min for equilibration, adsorption, and desorption cycles. The selectivity studies of the patulin imprinted and the non-imprinted SPR nanosensors are determined in the presence of ochratoxin A and aflatoxin B1. In order to demonstrate the applicability, validation studies of the patulin imprinted SPR nanosensor are performed by liquid chromatography-tandem mass spectrometry (LC-MS).