In this study, an associate analytical approach supported by high performance liquid chromatography (HPLC) was developed to analyze five isothiazolinones in liquid detergents in the meanwhile. The isothiazolinones were extracted from liquid detergents by ultrasound with methanol as the extraction solvent and analyzed by HPLC. The gradient elution procedure uses acetonitrile-water as the mobile phase. The isothiazolinones were effectively separated by Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 µm), and detected by diode array detector. The maximum absorption detection wavelengths of MIT, CMIT, OIT, DCOIT, and BIT were 275 nm, 275 nm, 282 nm, 285 nm, and 318 nm, severally. Quantitative analysis use of external standard method. The method was illustrated the excellent linearity with the standard curve correlation coefficients of determination (R2, 0.9992–1.0), LOD ranged from 0.06–0.19 µg/g. The spiked recoveries were 92.73%–109.92% with the RSD (n = 7) varied from 0.06%–2.26%. A total of 30 liquid detergents samples were inspected, the results demonstrated that MIT, CMIT, and BIT were contained with the concentration varied from 4.26 µg/g to 102.62 µg/g. And, it described the matrix effects of liquid detergents. Conclusion this method was recognized by excellent linearity, precision, accuracy, and was validated as a suitable and reliable technique for determination of isothiazolinone preservatives in liquid detergents.