The invention relates to sulphydryl single-element or multi-element organic acid surface-modified No.2-No.6 semiconductor quantum dots and a preparation method thereof. The method comprises the following steps of preparation of a precursor solution, preparation of quantum dots, and the like. The sulphydryl single-element or multi-element organic acid surface-modified No.2-No.6 semiconductor quantum dots are the quantum dots formed by CdX (X=S, Se, Te) or ZnX (X=S, Se, Te) which is coated by sulphydryl single-element (or multi-element) organic acid, wherein the mol ratio of the sulphydryl single-element (or multi-element) organic acid to the CdX or the ZnX is between 2:1 and 3:1; and particle diameter of the quantum dots is between 2.0 and 6.0 nanometers. The invention takes gas as a tellurium (or sulfur or selenium) source, and reduces impurity compositions of the precursor solution by introducing H2X (X=S, Se, Te); and due to the superior hydrophilicity of the sulphydryl single-element (or multi-element) organic acid, the prepared No.2-No.6 semiconductor CdX (X=S, Se, Te) and ZnX (X=S, Se, Te) quantum dots,, and the like have good dispersity and wide fluorescent range, and have the quantum yield as high as more than 80 percent. Simultaneously, the quantum dots have small toxicity, good biocompatibility and easy operation, and can be used for medicament tracking, diagnostic marking, and the like.