The adaptability of bacterial resistance to antibiotics contributes to its high efficiency during evolution. Tetracycline (TC) is a broad-spectrum antimicrobial agent. Chromatographic analyses and mass spectrometry were used to study the effects of the light illumination of a 462 nm light-emitting diode (LED) on the conformational changes of TC in a phosphate buffer solution (PBS, pH 7.8). Especially, the inactivation of superoxide anion radicals (O2•−) and Escherichia coli (E. coli), including that of a multidrug-resistant E. coli (MDR E. coli), were investigated during the photolysis of TC. A photolysis product of TC (PPT) was generated in an alkaline solution after the illumination of a blue light. The mass spectra of PPT had characteristic ion signals in m/z 459, 445, and 249.1 Da. The PPT has the molecular formula of C22H22N2O9, and the exact mass is 458.44 g/mol. The inactivation of MDR E. coli is not significant with TC treatment. The drug-resistant ability of MDR E. coli has a less significant effect on PPT, and the changed conformation of TC retained the inactivation ability of MDR E. coli upon blue light photoreaction. With TC, illuminated by a blue light in a pH 7.8 PBS, O2•− was generated from TC photolysis, which enhanced the inactivation of E. coli and MDR E. coli. A 96.6% inactivation rate of MDR E. coli was reached with TC under 2.0 mW/cm2 blue light illumination at 25 ± 3 °C for 120 min, and the effects of the TC-treated photoreaction on MDR E. coli viability repressed the growth of MDR E. coli by 4 to 5 logs. The present study of the blue light photoreaction of TC offers a new approach to the inactivation of MDR E. coli.