A flow-injection ultrafiltration sampling chemiluminescence system for on-line determination of cimetidinebovine serum albumin (BSA) interaction is proposed in this paper. Cimetidine can be oxidized by N-bromosuccinimide (NBS) and sensitized by fluorescein to produce high chemiluminescence emission in basic media. The concentration of cimetidine is linear with the CL intensity in the range 31071104molL1 with a detection limit of 1107molL1 (3). The drug and protein were mixed in different molar ratios in 0.067molL1 phosphate buffer, pH7.4, and incubated at 37C in a water bath. The ultrafiltration probe was utilized to sample the mixed solution at a flow rate of 5Lmin1. The data obtained by the proposed ultrafiltration flow-injection chemiluminescence method was analyzed with Scrathard analysis and a Klotz plot. The estimated association constant (K) and the number of the binding site (n) on one molecule of BSA by Scrathard analysis and Klotz plot were 3.15104Lmol1 and 0.95, 3.25104Lmol1 and 0.92, respectively. The proposed system proved that flow-injection chemiluminescence analysis coupled with on-line ultrafiltration sampling is a simple and reliable technique for the study of drugprotein interaction.