A flow-injection chemiluminescence CL.biosensor for the determination of total D-amino acid is described in this paper. D-amino acid oxidase was immobilized onto amine-modified silica gel via glutaraldehyde activation and packed in a column. The other analytical reagents, including luminol and ferricyanide, were electrostatically co-immobilized on an anion-exchange column. D-amino acid was sensed by the CL reaction between hydrogen peroxide produced from the enzyme column and luminol and ferricyanide, which were eluted from the ion-exchange column through sodium phosphate injection. The linear range of D-alanine concentration is 1.1×10-6 to 1.1×10-4mol/l and the detection limit was 4.5×10-7mol/l (3σ). The proposed method has been successfully applied to the determination of D-amino acid in human serum.