A fluorimetric method for the determination of vitamin B12 has been developed. The fluorescence emission was measured at lambda(ex)/lambda(em)275/305 nm in phosphate buffer solution (pH 7.0), and the experimental variables and possible interference were studied. The linear calibration range was 1.000 ng/mL to 100.0 ng/mL with a correlation coefficient of 0.9994 and a detection limit of 0.1 ng/mL. The method is rapid, simple and highly sensitive. It was used to determine vitamin B12 in pharmaceutical preparations. The recovery was 96%-98% and the relative standard deviation was in the range of 1.8%-2.7%. The results agreed with those obtained by spectrophotometry.