The invention provides a site-directed mutated rituximab containing unnatural amino acid markers and a method for conjugating the rituximab by site-directed mutation and site-directed micro-molecules. The method includes the steps: leading unnatural amino acid into rituximab genes by genetic codon extension technology in a site-directed manner; performing site-directed connection by the aid of the unnatural amino acid and modifiers such as poly-difunctional connecting arms DIBO-DOTA; further realizing site-directed conjugating of the rituximab and radioactive isotope such as Cu64 through DOBO-DOTA. The invention further relates to an application of site-directed conjugated micro-molecule rituximab as development, tracing and therapeutic radio-immunity conjugate.