A sensitive chemiluminescence method, based on the enhancive effect of phenobarbital on the chemiluminescence reaction between luminol and dissolved oxygen in a flow injection system, was proposed for the determination of phenobarbital. The chemiluminescence intensity responded to the concentration of phenobarbital linearly ranging from 0.05 to 10 ng⋅ml-1 with the detection limit of 0.02 ng⋅ml-1 (3σ). At a flow rate of 2.0 ml⋅min-1, a complete determination of phenobarbital, including sampling and washing, could be accomplished in 0.5 min, offering the sampling efficiency of 120 h-1 accordingly. The method was applied successfully in an assay of PB for pharmaceutical preparations, human urine and serum without any pretreatment with recovery from 95.7 to 106.7% and RSDs of less than 3.0%.