Chlortetracycline (CTC) is widely used as a veterinary antibiotic and is considered as a recalcitrant pollutant. In this study, spiked CTC (2 mg L-1) in wastewater was degraded using laccase from the white rot fungi, Trametes Versicolor combined with ultrasonication (UlS). Over 60% of CTC was removed in 2 h by UlS assisted laccase (UAL) treatment where laccase treatment alone took 2 days to degrade 87% of CTC under similar CTC concentration (2 mg L-1), laccase dose (0.5 IU) and pH 6.0 conditions. UAL treatment showed 5.3 folds higher CTC degradation rate compared to laccase alone treatment at pH 6.0. Further, pH optimization of UAL treatment was done and pH 4.5 was found to be optimum wherein 80% of CTC degradation was obtained which is 2.6 folds higher compared to degradation at pH 6.0. The UAL treatment with optimized pH was not only increased CTC degradation efficiency (similar to 80%) but also reduced the degradation time to 2 h. The obtained results highlighted the enhanced degradation rate, efficiency and unaltered stability of laccase during UAL treatment which can be used for oxidizing other tetracycline groups of antibiotics. Moreover, laccase and UAL treatments showed similar degradation products and no estrogenic activity.