The main purpose of this study was to develop an inexpensive, simple, rapid and sensitive chemiluminescence (CL) method for the determination of enrofloxacin (ENRO) using a flow-injection system. This method is based on rapid reduction of Ru(phen)(3)(3+), which is produced in the reaction between Ru(phen)(3)(2+) and acidic Ce(IV) by ENRO, producing strong CL. A central composite design (CCD) was used for optimization of the chemical variables. Regression analysis of the data from the CCD demonstrated that a second-order polynomial model is an adequate description of the surface over the factor limits studied. Optimization using CCD gave approximately four-fold better results than the single-factor-at-a-time method. Under optimal experimental conditions, the CL response was proportional to the concentration of ENRO over a wide range (0.008-3.6 microg/mL) with a correlation coefficient of 0.9986 and a detection limit of 0.003 microg/mL (3sigma). The relative standard deviation for 11 repeated determinations of 0.14 microg/mL ENRO was 4.2%. This method was successfully applied to the analysis of commercial formulations, spiked plasma and spiked poultry tissue. Sample analyses showed good recovery percentages for drugs and spiked plasma (95.1-103.9%). Recovery percentages for spiked poultry tissue were in the range 77.6-87.3%. The minimum sampling rate was 100 samples/h.