A novel fluidized beads strategy for utilization of particle-immobilized reagents in flow analysis was developed in this study. The performance of the suggested strategy was demonstrated by the determination of glycerol in biodiesel. This analytical task was used as a proof-of-concept example. The method is based on on-line extraction of glycerol from biodiesel into aqueous stationary phase of extraction-chromatographic column, followed by elution and spectrophotometric determination in the form of copper glycerate formed in a fluidized reactor of stepwise injection system. The floating of cation exchange resin Dowex^® 50WX4, saturated with Cu(II) ions in liquid phase, was accomplished by air-bubbling. The linear range was from 100 to 1000 mg kg^-1, and the limit of detection, calculated as 3s of a blank test (n = 5), was found to be 30 mg kg^-1. The method was successfully applied to the analysis of biodiesel and biodiesel-blend (B 20) samples.