In the current article, chemiluminescence (CL) from the vitamin B 12 and luminol reaction was studied under alkaline conditions to develop a sensitive analytical method for vitamin B 12 using the carbonate enhancement effect. The method was successfully applied to the determination of vitamin B 12 in vitamin B 12 tablets, multivitamin capsules, and vitamin B 12 injections. Experimental parameters were optimized, including luminol concentration, urea–hydrogen peroxide (urea–H 2 O 2 ) concentration, effect of pH, and sequence of addition of reactants for obtaining maximum CL, which was not explored previously. The limit of detection was 5pg/ml, and the linear range was 10pg/ml to 1μg/ml with a regression coefficient of R 2 =0.9998. The importance of these experimental parameters and the carbonate enhancement effect is discussed based on the knowledge of the mechanism of oxidation of luminol and decomposition of urea–H 2 O 2 in the presence of vitamin B 12 . Extraction of vitamin B 12 was carried out, and the observed recovery was 97–99.2% with a relative standard deviation in the range of 0.30–1.09%. The results obtained were compared with those of the flame atomic absorption spectrometry method.