Biodiesel is an important alternative fuel produced from animal fats or vegetable oils. According to several world regulatory agencies, the acceptable threshold content for free glycerol in biodiesel is 200 mg kg-1. Exceeding concentrations may yield high amounts of acrolein after combustion. The analytical methods for glycerol determination are usually preceded by conventional liquid-liquid extraction that may affect precision and accuracy and impair analytical frequency. In this work, a multi-pumping flow system was proposed for the online dispersive liquid-liquid extraction of free glycerol from biodiesel followed by spectrophotometric determination. The analyte was transferred to the aqueous phase by mixing the sample and water under a pulsed flow regime. The emulsion was directed towards a retention column to remove the organic phase before chemical derivatization. Glycerol was oxidized by NaIO4 to generate formaldehyde, which reacted with acetylacetone in an ammonium acetate medium to yield 3,5-diacetyl-1,4-dihydrolutidine (λMAX = 412 nm). The optimization of the main parameters of the system was carried out by multivariate methods. The screening of variables was performed by fractional factorial design 24-1. The models for free glycerol determination and extraction were refined by central composite and full factorial 23, respectively. In both cases, the validation was carried out by analysis of variance, which yielded a satisfactory F-test value. After optimization, a linear range from 3.0 to 50.0 mg L-1 glycerol was observed. The detection limit, coefficient of variation and determination frequency were estimated to be 2.0 mg L-1 (n = 20; 99.7% confidence level), 4.2-6.0% (n = 20) and 16 h-1, respectively. The efficiency of the process was estimated to be 66%. After each extraction, the retention column (filled with 185 mg of glass microfiber) was washed with 50% ethanol solution to avoid carry-over effects. The comparative analyses of samples by the proposed and reference methods demonstrated the accuracy of the developed procedure at a 95% confidence level. Recoveries between 86 and 101% also indicated that the proposed procedure is accurate, suitable and reliable for online extraction and determination of free glycerol in biodiesel.