An ultrasensitive and selective light-scattering (LS) aptasensor for prostate specific antigen (PSA) was developed based on bifunctional DNA decorated gold nanoparticles (Au NPs) by use of target stimuli-responsive assembly. Both binding sequence poly adenine (poly(dA), Domain I) and the recognition sequence aptamer (Domain II) of bifunctional ssDNA were in favor of the sensitivity for the fabricated light-scattering aptasensor due to modulated the lateral spacing of DNA on Au NPs surface and possessed strong antigen binding affinity, respectively. And they efficiently promoted the Au NPs aggregation and activated the signal of light-scattering of aptasensor. The formation of aptamer-PSA complex induced conformational transformation of the aptamer on the surface of the Au NPs-based aptasensor and then tuned the interparticle distance of gold nanoparticle assemblies. The larger scattering particles, the higher light-scattering intensity. Target stimuli-responsive aggregation behavior of Au NPs lit up the light-scattering signal of aptasensor. As the concentration of PSA increased, the light-scattering intensity gradually enhanced. The aptasensor response for PSA detection was in the linear range from 0.01 ng/mL to 20 ng/mL with the detection limit of 2 pg/mL. The thiol-free Au NPs-based light-scattering aptasensor has exceptional performances with ultrasensitivity and high selectivity besides the advantage of economic and facile fabrication. Moreover, the proposed target-activated aptasensor was applied for the detection of PSA in serum samples and demonstrated great potential in clinical applications.