In the present study, a simple and sensitive chemiluminescence method using tris(2,2′-bipyridyl)ruthenium(III) with flow injection detection is reported for the analysis of trimethoprim in pharmaceutical preparations. The calibration graph was linear over the concentration range of 0.05- 5.0 µg mL-1 with relative standard deviations (n = 4) in the range 1.0 – 2.5%. The limit of detection (3s blank) was 0.008 µg mL-1 with a sample throughput of 100 h-1. The effect of some organic compounds, common excipients used in pharmaceutical preparations, cations & anions were studied for trimethoprim determination. Ru(bipy)32+  was chemically oxidized to Ru(bipy)33+ online using lead dioxide mini column.