Although phosphate (Pi) is a necessary nutrient for the growth of aquatic organisms, the presence of excess Pi leads to water eutrophication; thus, it is necessary to accurately determine the content of Pi in water. A method for the determination of trace Pi in aquaculture water was developed based on surface-enhanced Raman spectroscopy (SERS) combined with rhodamine 6G (R6G)-modified silver nanoparticles (AgNPs) as the active substrate. The adsorption of R6G on the AgNP surfaces led to a strong SERS signal. However, in the presence of Pi and ammonium molybdate, phosphomolybdic acid formed, which further associated with R6G to form a stable R6G-PMo₁₂O₄₀³⁻ association complex, thereby hindering the adsorption of R6G on the AgNPs, and reducing the SERS intensity; this sequence formed the basis of Pi detection. The decrease in the SERS intensity was linear with respect to the Pi concentration (0.2–20 μM), and the limit of detection was 29.3 nM. Upon the application of this method to the determination of Pi in aquaculture water, a recovery of 94.4–107.2% was obtained (RSD 1.77–6.18%). This study provides an accurate, rapid, and sensitive method for the trace determination of Pi in aquaculture water, which is suitable for on-site detection.