Biochar (BC) and rhamnolipid (RL) is used in bioremediation of petroleum hydrocarbons, however, the combined effect of BC and RL in phytoremediation has not been studied until now. In this paper, the phytoremediation of petroleum hydrocarbon-contaminated soil using novel plant Spartina anglica was enhanced by the combination of biochar (BC) and rhamnolipid (RL). Samples of petroleum-contaminated soil (10, 30 and 50 g/kg) were amended by BC, BC + RL and rhamnolipid modified biochar (RMB), respectively. After 60 day's cultivation, the removal rate of total petroleum hydrocarbons (TPHs) for unplanted soil (UP), planted soil (P), planted soil with BC addition (P-BC), planted soil with BC and RL addition (P-BC + RL) and planted soil with addition of RMB (P-RMB) were 8.6%, 19.1%, 27.7%, 32.4% and 35.1% in soil with TPHs concentration of 30 g/kg, respectively. Compared with UP, the plantation of Spartina anglica significantly decreased the concentration of C8–14 and tricyclic PAHs. Furthermore, the application of BC and RMB alleviated the toxicity of petroleum hydrocarbons to Spartina anglica via improving plant growth with increasing plant height, root vitality and total chlorophyll content. High-throughput sequencing result indicated that rhizosphere microbial community of Spartina anglica was regulated by the application of BC and RMB, with increase of bacteria and plant mycorrhizal symbiotic fungus in biochar and RMB amended soil.