Due to the high sensitivity and real-time detection, surface plasmon resonance microscopy (SPRM) has been widely used in nano-detection, biomedicine, and environmental monitoring. As the evanescence wave propagates along the interface, a special point spread function of SPRM is formed, from which rich information of the analyte can be retrieved. However, the defocus can affect the imaging pattern, which hinders the acquisition of accurate analyte information. As a result, the quantitative study on the effect of defocus on SPRM is crucial. This study quantitatively analyzes the effect of defocus on SPRM both theoretically and experimentally, and realizes the SPRM imaging of single polystyrene nanoparticles. The proposed method can be used to rapidly judge the defocus status of SPRM, retrieve the accurate defocus displacement to achieve fast re-focusing, and improve the SPRM performance in long-term observation.