Abstract: HPLC was applied to the determination of 5 fluorescent whitenings in cotton fabric with ultrasonic extraction of sample. The sample was extracted ultrasonically with DMF-H2O (70+30) mixture for 20 min at 40 ℃, and the extract was separated on Agilent Eclipse XDB-C18 chromatographic column with mixtures of acetonitile and 10 mmol·L-1 ammonium acetate solution with different ratios as mobile phase in the gradient elution, and detected with DAD. Linear relationships between values of peak area and mass concentration of the 5 fluorescent whitenings were kept in the same range of 0.5-200.0 mg·L-1, with detection limits (3S/N) in the range of 0.05-0.10 mg·L-1 and lower limits of determination (10S/N) in the range of 1.2-2.1 mg·kg-1. On the base of blank sample, test for recovery was made by standard addition method;values of recovery found were in the range of 76.5%-103%, with RSD′s (n=6) in the range of 2.6%-7.1%.