Sulfamethoxazole (SMX) is a sulfonamide antibiotic primarily used to treat urinary tract infections and used in veterinary and industrialized husbandry to treat diseases and food additives. Like other antibiotics, SMX is considered as a pollutant in water and food that threaten local life. This study developed a surface plasmon resonance (SPR) sensor chip that is fast, highly selective, and reusable, and requires no pretreatment for detecting SMX. As a receptor, SMX imprinted methacrylic acid-2-hydroxyethyl methacrylate-ethylene glycol dimethacrylate polymer [poly(MAA-HEMA-EGDMA)] was used. The surface of the gold SPR chips was coated with a drop-casting method. The nanofilm coated chips were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), ellipsometer, contact angle measurement, and Fourier-transform infrared spectrometry (FTIR). Imprinting factor (IF) was calculated as: ΔR[MIP(molecularly imprinted polymers)]/ΔR[NIP(non-imprinted)]=12/3.5=3.4. Limit of detection (LOD) and limit of quantification (LOQ) values were calculated with 3 s/m and 10 s/m methods, and the results were found to be 0.001 1 μg/L for LOD 0.003 4 μg/L for LOQ. Adsorption studies on both standard SMX solution and commercial milk samples were applied. Also, we investigated the developed chip’s reusability, storability, and selectivity with amoxicillin and cefalexin.