A high-performance liquid chromatography (HPLC) method with fluorescence detection is described for the determination of sodium monofluoroacetate (MFA-Na) in biological samples. 4-Bromomethyl-7-methoxycoumarin is used as a derivatization reagent and reacted with MFA-Na to form 7-methoxy-4-methylenecoumarin monofluoroacetate for HPLC analysis. Chromatographic separation is performed on a Hewlett Packard RP-18 column using methanol-water (60:40, v/v) as the mobile phase. A fluorescent detector is employed with the excitation and emission wavelengths as 319 nm and 390 nm, respectively. The novel method yields a good linear relationship when the concentration of MFA-Na is within 1 and 500 nmol/mL (r = 0.9996). The detection limit is 50 pmol/mL. The established method is applied to determine MFA-Na in biological samples. The recovery rates of MFA-Na are between 81% and 88%, and the relative standard deviations are less than 5%. The method shows good sensitivity and selectivity for the determination of MFA-Na in biological samples.