An ultrasensitive and highly selective testosterone detection method by surface plasmon resonance (SPR) combined with molecularly imprinted films (MTFs) was developed. The coupling angle shifts were highly selective to testosterone and showed a linear relationship to the logarithmic concentrations (from 10(-15) to 10(-10) mol L-1) of the testosterone in the acetonitrile. The selectivity of the thin testosterone-imprinted films was examined by 10(-4) mol L-1 progesterone acetonitrile solution, no observable binding was detected.