A sequential injection procedure involving a flow-reversal wetting-film extraction method for the determination of the radionuclide 90Sr has been developed. The methodology is based on the coating of the inner walls of an open tubular reactor with a film prepared from a 0.14 M 4,4'(5')-bis(tert-butylcyclohexano)-18-crown-6 (BCHC) solution in 1-octanol, which allows the selective isolation of strontium from the sample matrix. Selection of the optimum extractant diluent attending its physical properties, investigation of the extraction kinetics features, and choice of the proper elution procedure are discussed in detail in this paper. The noteworthy aspects of using a wetting-film phase instead of a solid-phase material described to date in the literature are the reduction of crown ether consumption and the simplification of both the operational sequence and the automation of the extractant-phase renewal between consecutive samples, which is of interest to avoid analyte carryover and reduction of the resin capacity factor caused by irreversible interferences. The proposed method has been successfully applied to different spiked environmental samples (water, milk, and soil), with 90Sr total activities ranging between 0.07 and 0.30 Bq, measured using a low-background proportional counter. The standard deviation of the automated analytical separation procedure is lower than 3% (n = 10), and the 90Sr isolation process under the studied conditions may be carried out with a yield up to 80%.