In this study, an ultrasensitive surface-enhanced Raman scattering (SERS) detection of alkaline phosphatase (ALP) has been developed, in which nile blue A (NBA) was chosen to replace nitro blue tetrazolium chloride (NBT) in a reactive system of 5-bromo-4-chloro-3-indolyl phosphate (BCIP), NBT, and ALP. In the reactive process, NBA was converted to a low SERS-active molecule, and its SERS intensity at 592 cm⁻¹ decreased when NBA was reduced by 5-bromo-4-chloro-3-indolyl, which converted from BCIP by ALP. The SERS signal of NBA was inversely proportional to the amount of ALP in the reactive system. Based on convenient SERS materials of gold nanoshells absorbed on acupuncture needles, the detectable concentration range of ALP was 1–10⁴ mU L⁻¹ in a reactive system of BCIP, NBA, and ALP.