Anti-solvent crystallization of potassium dihydrogen phosphate (KDP) from aqueous solution was studied by using on-line Raman spectroscopy. The used anti-solvent was acetone and semi-batch crystallization experiments were conducted by adding acetone into an initially pure aqueous KDP solution saturated at 25 °C. The solubility of KDP in acetone-water mixtures was determined at 25 °C. A calibration model of Raman spectra was created using a series of KDP-water-acetone ternary or binary solutions, and the calibration model was used to attain the on-line KDP concentration profile during the crystallization. The evolution of supersaturation during the whole process was then obtained. The reliability of the calibration model was validated by the off-line measured KDP concentrations. The effects of the feeding rate and feeding position of the anti-solvent on precipitation were discussed based on the obtained supersaturation profile.