Biochar is investigated experimentally as a new highly effective amendment to remediate contaminated soil. A crucial consideration is the influence of biochar on the bioremediation of soil polluted with total petroleum hydrocarbons (TPHs), and in particular, the use of biochar as a bacteria immobilization carrier with a synergistic effect of absorption and degradation. Therefore, we studied the ability of petroleum-degrading bacteria immobilized on biochar, free bacteria, and biochar alone on the removal of TPHs in soil using gravimetric analysis and gas chromatography-mass spectrometry. After 60 days of remediation, the strategy involving immobilized bacteria on biochar was more effective than other treatments in reducing the contents of TPHs and n-alkanes with C12-18, which showed the shortest half-life and highest biodegradation efficiency; variations in the features of enzymatic activities and microbial respiration indicated that the biochar treatment improved not only the soil fertilizer and carbon storage, but the immobilization greatly affected both the physicochemical properties of soil and bacterial activities. Moreover, the bacterial population diversity and bioavailability of hydrocarbons were promoted by the inputs of the combination of biochar and petroleum-degrading bacteria. Overall, our results highlight the potential of applying immobilized microorganisms on biochar for accelerating the biodegradation of petroleum and maintaining the balance of the soil ecosystem, which may be ascribed to the synergistic effect of biostimulation and bioaugmentation.