A new, rapid flow injection (FI) method is reported for the spectrophotometric determination of monofluorophosphate (MFP) ions in toothpastes. MFP ions are hydrolyzed on-line by alkaline phosphatase (APase) immobilized on a cellulose nitrate membrane, prior to injection in the FI system. The yielded orthophosphate ions are determined spectrophotometrically (λ_max = 690 nm) using the molybdenum blue approach. The chemical and FI variables that affected the enzymatic reaction were studied and optimized. A study of interferences was also carried out. The proposed method is very precise (s_r = 0.7% at 1.0 × 10⁻⁴ mol l⁻¹ MFP, n = 12), fast (sampling rate of 72 h⁻¹) and allows the determination of MFP ions in the range of 4.0 × 10⁻⁵ to 6.0 × 10⁻⁴ mol l⁻¹ with a satisfactory 3σ detection limit of 4.0 × 10⁻⁶ mol l⁻¹. The application of the proposed FI method to toothpaste samples yielded accurate results (e_r < 2.0%) compared with a potentiometric reference procedure.