The interaction of crystal violet (CV) with yeast RNA (yRNA) was studied by UV-Vis absorption spectrophotometric method in this paper and further a simple spectrophotometric method for the yRNA determination was proposed. At a pH 4.0 Britton-Robinson (B-R) buffer solution, CV had a máximum absorption peak at 590 nm, which was greatly decreased by the addition of yRNA. The conditions of CVinteracted with yRNA were carefully investigated. Underthe optimal conditions the decrease of absorbance value was proportional to the concentration of yRNA in the range from 3.33x10-6 to 1.67x10-5 mol L-1 when 2.0 x 10(5) mol L-1 CV was employed. The detection limit was calculated as 5.34 x 10-7 mol L-1 (3σ) and different samples were determined satisfactorily. The binding number and the binding constant of CV with yRNA were calculated by spectrophotometric data with the results as 1 and 1.69 x 10(5) L mol-1, respectively. The reaction mechanism was discussed with the electrostatic attraction led to molecular association of yRNA with CV