•MAE-SPP-DLLME was developed for the analysis of antibacterial drugs in fish samples.•Experimental conditions of MAE were optimized through orthogonal array design.•Less matrix effects with MAE-SPP-DLLME compared to MAE-SPP.A novel pretreatment method involving microwave-assisted extraction and solid-phase purification combined with dispersive liquid–liquid microextraction (MAE-SPP-DLLME) followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) was established for the simultaneous determination of six antibacterial pharmaceuticals including metronidazole, tinidazole, chloramphenicol, thiamphenicol, malachite green and crystal violet. The conditions of MAE were optimized using an orthogonal design and the optimal conditions were found to be 8 mL for acetonitrile, 50 °C for 5 min. Then, neutral alumina column was employed in the solid-phase purification. Finally, the critical parameters affecting DLLME, including selection of extraction and dispersive solvent, adjustment of pH, salt concentration, extraction time, were investigated by single factor study. Under optimum conditions, good linearities (r > 0.9991) and satisfied recoveries (Recoveries > 87.0%, relative standard deviation (RSD) < 6.3%) were observed for all of the target analytes. The limits of detection and quantification were 4.54–101.3 pg kg−1 and 18.02–349.1 pg kg−1, respectively. Intra-day and inter-day RSDs were all lower than 3.6%. An obvious reduction in matrix effect was observed by this method compared with microwave assisted extraction followed by purification. The established method was sensitive, rapid, accurate and employable to simultaneously determine target analytes in farmed fish, river fish and marine fish.