A novel ultrasound-assisted micellar cleanup strategy (UAMC) coupled with large volume injection (LVI) high performance liquid chromatography (HPLC) method was proposed and successfully applied to the analysis of cefathiamidine in complex biological samples such as whole blood, plasma, serum and even zebrafish, a challenging positive real sample. Based on the micelle-biomacromolecule interaction, the phase-separation feature of surfactant micelles and ultrasound cavitation, UAMC possessed an impressive matrix cleanup capability and could rapidly reach distribution equilibrium (approximately 2 min), which enabled simultaneous sample cleanup and analyte extraction within 8 min. Due to the high cleanup efficiency of UAMC, large volume of pretreated samples could be injected for analysis without peak broadening, impurity interference and column degradation. Thus, online analyte enrichment could be automatically performed to significantly improve method sensitivity by the column-switching LVI-HPLC system, a commercial HPLC system with small modifications. The UAMC-LVI-HPLC method creatively integrated sample cleanup, analyte extraction and on-column enrichment into simple operation. In addition, the UAMC-LVI-HPLC method enabled non-matrix-matched analysis of cefathiamidine in complex biological samples. This feature was helpful to address the problems caused by conventional matrix-matched or internal standard calibration methods, such as matrix bias, increased workload, limited availability of suitable blank matrices and the use of expensive internal standards. The method had low limits of detections (e.g., 0.0051 mg/L and 0.038 μg/g), wide linear ranges (0.030-100 mg/L and 0.15-489 μg/g), good linear correlation (R2 = 0.9999), satisfactory accuracy (97.6-109.7%) and excellent intra- and interday precision (0.5-4.9%). Thus, UAMC-LVI-HPLC is expected to be a promising candidate for bioanalysis in therapeutic drug monitoring or pharmacokinetic and toxicology studies in the future.