The aim of the present work was to develop a new method using synchronous fluorescence spectrometry (SFS) to determine the concentration of isoliquiritigenin (ISL) in mouse blood and tissues, and to investigate ISL's distribution among organs after an intraperitoneal (IP) dose of ISL. The synchronous fluorescence method was optimized with the sample pH, stability, metal ions, concentration of Al3+, and surfactants. The proposed method was used to determine the ISL concentration in mouse blood, brain, heart, kidney, liver, spleen and lung after an IP injection of ISL. The optimal conditions for the determination of ISL using SFS were found to be: excitation and emission wavelengths of 469 and 5.57 nm, respectively; the use of 3% AlCl3 as a fluorescence intensity enhancer; measuring samples within 1 h of collection, sample pH 7 - 8, isolation of samples from surfactants; and wavelength interval (Delta lambda) = 70 nm. After IP injection, the distribution of ISL in mouse organs was: liver > kidney > spleen > blood > lung > brain > heart. The blood concentration of ISL, peaked at 60 min; concentrations of ISL in liver, kidney and spleen achieved maxima at 120 min. SFS provides a simple, but effective analytical method that will benefit the study of in vivo biological effects of ISL, including absorption, distribution, metabolism, and excretion.