The selective, sensitive, and real‐time detection of testosterone was performed from both testosterone aqueous solution and artificial urine samples by using the advantages of surface plasmon resonance sensor (SPR) with molecular imprinting technique. The testosterone imprinted (MIP) and non‐imprinted (NIP) nanoparticle based poly(2‐hydroxyethyl methacrylate‐methacrylic acid (poly(HEMA‐MAA)) SPR sensors were prepared for detection of testosterone. After characterization of MIP and NIP nanoparticles, nanoparticles were attached on the SPR chip surfaces. The characterization of SPR sensor surfaces was carried out with characterization methods such as atomic force microscopy, ellipsometer and contact angle measurements. The real‐time measurements for detection of testosterone were performed with testosterone imprinted SPR sensor in the linear range of 0.5‐20 ng/mL concentration in testosterone aqueous solution. The imprinting factor and the limit of detection was found 4.16 and 0.049 ng/mL, respectively. The validation studies were performed with both SPR sensor and enzyme‐linked immunosorbent assay using testosterone samples prepared in artificial urine samples for the detection of testosterone.