Levofloxacin, as a typical fluoroquinolone antibiotic, is widely used in human bacterial infection treatment and animal husbandry. However, conventional treatment technologies (e.g., precipitation, coagulation, and microbial metabolism) have limited ability to remove levofloxacin from wastewater. In this work, Fenton oxidation combined with ultrasonic treatment was investigated to efficiently remove levofloxacin from water. As a result, the optimal conditions for Fenton oxidation were first determined by the design of an orthogonal experiment. Then the combined effect of Fenton oxidation and ultrasonic treatment demonstrated a positive synergistic effect. In terms of the sequence selection of Fenton oxidation and ultrasonic treatment, Fenton–ultrasonic (10 min)–interval (40 min) was the best process. Finally, a total of 12 intermediates of levofloxacin were identified via HPLC-MS spectra, and possible degradation pathways were tentatively inferred. In addition, the toxicities of the intermediates were estimated using the Toxicity Estimation Software Tool according to the U.S. EPA proposed standards, and the results suggest that the overall toxicities of the intermediates were relatively alleviated in comparison with levofloxacin. This study provides a strategy for improving the degradation of levofloxacin via Fenton oxidation combined with ultrasonic treatment with a positive synergistic effect in water.