In this study, 17 beta-estradiol (E2) was performed by surface plasmon resonance (SPR) based on molecularly imprinted technique. In the first, SPR sensor was prepared by the formation of E2 imprinted poly(2-hydroxyetyl methacrylate) / (N-methacryloyl-(L)- leucine methyl esters) (PHEMALM) on the modified gold surface of SPR chip. Also, non-imprinted SPR chip was prepared by the same method at the E2 imprinted PHEMALM (E2-MIPHEMALM-SPR) chip without E2. E2-MIPHEMALM-SPR and non-imprinted (NIPHEMALM-SPR) chips were characterized with Fourier transform infrared spectrophotometry (FTIR), ellipsometry, atomic force microscopy (AFM) and contact angle measurements. After that, SPR chips were completed to SPR system to investigate kinetic properties for E2. The sensing ability of E2-MIPHEMALM-SPR chip was investigated with 20-10000 ng/mL concentrations of 17 beta-estradiol solutions. The E2-MIPHEMALM-SPR chip was showed more selectivity for 17 beta-estradiol than NIPHEMALM-SPR chip. To show the selectivity of E2-MIPHEMALM-SPR chip competitive adsorption of 17 beta-estradiol, cholesterol and stigmasterol were investigated. E2-MIPHEMALM-SPR chip was investigated ten times with same concentrations of 17 beta-estradiol solution to show reuse of the chip. The results showed that the E2-MIPHEMALM-SPR chip has high selectivity for 17 beta-estradiol.