The purpose of this study is to assess Oxolinic acid residues in grouper (Epinephelus coioides) and black seabream (Acanthopagrus schlegelii) and generate a basis for setting withdrawal periods, which could be used for reference in regulating newly approved aquatic animal drugs and usage policies. Based on the test methods announced by the Ministry of Health and Welfare, residues of Oxolinic acid in fish tissue was analyzed by ultra-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UPLC–MS/MS) under the multiple reaction monitoring (MRM) mode. Under the optimal conditions, the linear range was 5-1000 μg/L, the results showed a good linear relationship (R2= 0.9972). The limit of detection (LOD) was 3 μg/L and the limit of quantitation was 5 μg/L. The spiked levels were 10, 50, 100 μg/kg. The average recoveries of spiked test in grouper were 79.0-81.4 % in muscle with skin and 82.6-88.5 % in liver. The spiked test in black seabream, the average recoveries were 84.8-92.6 % in muscle with skin and 91.8-99.3 % in liver. The intra-assay of the muscle (with skin) and liver relative standard deviation (RSD) in grouper was 3.8-9.2 % and 5.4-8.5 % respectively, and the inter-assay was 1.5-3.2 % and 2.3-6.4 %. The intra-assay of the muscle (with skin) and liver RSD in black seabream was 1.4-5.2 % and 4.2-5.6 % respectively, and the inter-assay was 1.5-2.5 % and 2.2-3.2 %, respectively. The residual test was administered orally into two doses 30 and 60 mg/kg, for five consecutive days. The samples were collected at 1st, 3rd, 5th, 7th, 10th, 14th and 21st day,. The results showed that the residue levels in the muscles and livers with low and high doses were both under the method detection limit (3 μg/kg) on the 5th day in black seabream and the 10th day in groupers. Therefore, the withdrawal time is estimated to be 8 days in black seabream and 15 days in groupers.