A novel chemiluminescence (CL) flow injection method for the determination of balofloxacin is described. The method is based on the weak CL signal arising from the reaction of $KBrO_3$ with $Na_2S_2O_4$ in acidic medium being significantly enhanced by balofloxacin in the presence of europium (III) ion and sodium dodecyl benzene sulfonate (SDBS). The experimental conditions that affected CL intensity were carefully optimized and the CL reaction mechanism was briefly discussed. Under the optimum conditions, the relative CL intensity was proportional to the concentration of balofloxacin in the range of $7.0{ imes}10^{-11}$ to $3.0{ imes}10^{-7}g;mL^{-1}$. The detection limit was $2.7{ imes}10^{-11}g;mL^{-1}$ and the relative standard deviation was 2.1% for $7.0{ imes}10^{-10}g;mL^{-1}$ balofloxacin (n = 13). The proposed method was successfully applied to the determination of balofloxacin in pharmaceutical formulations and biological fluids.