In this study, we used the surface stabilizer 3‐mercaptopropionic acid (MPA) to synthesize ZnSe quantum dots in nontoxic aqueous solvents. Because of the –COOH group, MPA is an SH‐R‐COOH molecule that is added to improve the dispersion and quantum dot (QD) compatibility with antibodies. The different structures ZnSe, Mn‐doped ZnSe, Mn‐doped ZnSe/ZnS, and ZnSe/Mn‐doped ZnS/ZnS were investigated to find the optimum photoluminescence (PL). The structure and distribution element of ZnSe/Mn‐doped ZnS/ZnS QD were analyzed by the high‐resolution TEM image and scanning transmission electron microscopy. This finding of the ZnS buffer layer helps to reduce the defect lattice of QDs, which increases PL significantly. The highest PL intensity of ZnSe/ZnS:Mn/ZnS is significantly more than that of ZnSe and ZnSe/ZnS 6.2 and 3.3 times, respectively. In addition, the fluorescence efficiency of ZnSe/ZnS:5%Mn/ZnS‐MPA was 74.37% higher than that of ZnSe:5%Mn/ZnS‐MPA (55.04%) in ZnS buffer.