In-situ chemical oxidation with persulfate (PS-ISCO) is a preferred approach for the remediation of fuel-contaminated groundwater. Persulfate (PS) can be activated by various methods to produce stronger sulfate radicals for more efficient ISCO. Despite karst aquifers being widespread, there are few reports on PS-ISCO combined with Fe2+-activated PS. To better understand the effects of Fe2+-activated PS for the remediation of gasoline-contaminated aquifers in karst areas, a box-column experiment was conducted under flow conditions, using karst groundwater and limestone particles to simulate an aquifer. Gasoline was used as the source of hydrocarbon contaminants. Dissolved oxygen and nitrate were added to enhance bioremediation (EBR) and ferrous sulfate was used to activate PS. The effect of Fe2+-activated PS combined with biodegradation was compared during the periods of EBR + ISCO and ISCO alone, using the mass flow method for data analysis. The results showed that the initial dissolution of benzene, toluene, and xylene (BTX) from gasoline injection was rapid and variable, with a decaying trend at an average pseudo-first-order degradation rate constant of 0.032 d−1. Enhanced aerobic biodegradation and denitrification played a significant role in limestone-filled environments, with dissolved oxygen and nitrate utilization ratios of 59 ~ 72% and 12–70%, respectively. The efficiency of EBR + ISCO was the best method for BTX removal, compared with EBR or ISCO alone. The pseudo-first-order degradation rate constants of BTX reached 0.022–0.039, 0.034–0.070, and 0.027–0.036 d−1, during the periods of EBR alone, EBR + ISCO, and ISCO alone, respectively. The EBR + ISCO had a higher BTX removal ratio range of 71.0 ~ 84.3% than the ISCO alone with 30.1 ~ 45.1%. The presence of Fe2+-activated PS could increase the degradation rate of BTX with a range of 0.060 ~ 0.070 d−1, otherwise, with a range of 0.034–0.052 d−1. However, Fe2+-activated PS also consumed about 3 times the mass of PS, caused a further decrease in pH with a range of 6.8–7.6, increased 3–4 times the Ca2+ and 1.6–1.8 times the HCO3− levels, and decreased the BTX removal ratio of ISCO + EBR, compared to the case without Fe2+ activation. In addition, the accumulation of ferric hydroxides within a short distance indicated that the range of PS activated by Fe2+ may be limited. Based on this study, it is suggested that the effect of Fe2+-activated PS should be evaluated in the remediation of non-carbonate rock aquifers. [ABSTRACT FROM AUTHOR]