In clinical diagnosis, the level of biological enzymes in serum has been generally regarded as markers of human diseases. In this work, a kind of simple and sensitive plasmonic probe (indicated as Au@FeOOH) has been synthesized with the guidance of plasmonic imaging and subsequently developed for the alkaline phosphatase (ALP) level detection under dark-field microscopy (DFM). As a kind of hydrolysis enzyme, ALP can promote the hydrolysis of l-ascorbic acid 2-phosphate to ascorbic acid (AA). AA further acts as a strong reduction reagent for the decomposition of the FeOOH shell, which results in a blue shift of localized surface plasmon resonance spectra and an obvious color change under DFM. RGB analyses show that using a ΔR/G value instead of scattering wavelength or R/G value as the analytical signal, the deviation attributed to the size distribution of the initial Au NPs is greatly suppressed, and a linear range from 0.2 to 6.0 U/L (R2 = 0.99) and a limit of detection of 0.06 U/L are acquired with various concentrations of ALP during the detection. Besides, this approach exhibits excellent selectivity in complex biological serum samples, which is expected to be applied for the early diagnosis of clinical diseases by monitoring various biomarkers in the future.