Objective: A HPLC method for the determination of anthocyanins in fruits of Lycium ruthenicum Murr. was developed. Methods: A Kromasil C18 (4.6 mm × 250 mm, 5 μm) column was used as the chromatographic column and a mixture of 0.2% HCOOH-H2O and acetonitrile (98:2) was used as the mobile phase at a flow rate of 1 mL/min for gradient elution. UV detection wavelength was set up at 520 nm. MAL standards were used for calibration. Results: An excellent linear relationship was achieved in the range of 43–935 μg/mL with correlation coefficient of 0.9994 and RSD of 2.73%. The recovery rate of in spiked samples was 101.01% with RSD (n = 6) of 2.12%. Conclusion: This developed method is simple, stable, fast, and reproducible and without the interference of impurities, so that it can be used for determining the content of anthocyanins in fruits of Lycium ruthenicum Murr..