The paper presents a novel method for the flotation separation of Bi3+ using microcrystalline adsorption system prior to the determination by spectrophotometry. The effects of different parameters, such as the dosages of KI and dodecyl trimethyl ammonium bromide (DTMAB), various salts and acidity etc. on the flotation yield of Bi3+ have been investigated to select the optimum experimental conditions. The possible flotation separation mechanism of Bi3+ was discussed. The results showed that under the optimum conditions, dodecyl trimethyl ammonium bromide cation (DTMAB+ ) reacted with I− to produce themicrocrystalline matter (Ms‐M) of (DTMAB+·I−), the water‐insoluble ternary association complex of (DTMAB)3[BiI6] which produced by Bi3+ and I−, DTMAB+ was quantificationally adsorbed on the surface of Ms‐M of (DTMAB+·I−) and was floated above water phase, the liquid‐solid phases were formed with clear interface. In this condition, K+, Na+, Ca2+, Mg2+, Co2+, Ni2+, Mn2+, Zn2+, Fe2+ and Al3+ could not be floated. Therefore, Bi3+ was separated completely from the above metal ions. A new method for flotation separation and determination of bismuth using microcrystalline adsorption system was established. The proposed method has been successfully applied to the determination of Bi3+ in bismuthiferous drug samples, and the results agreed well with those obtained by pharmacopoeia method. The recoveries were 94.1%∼106.8%, and the RSD was 1.5%∼2.3%.