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The use of trastuzumab as intact IgG labeling radionuclide for HER2 positive breast cancer theranostic agent is not ideal because it is slowly eliminated from the blood and normal tissues resulting in low tumor/blood (T/B) and tumor/normal tissue (T/NT) ratios. To overcome this limitation, we developed the trastuzumab F(ab′)2 fragments and radiolabeling of the fragments by β and γ-particle of Lutetium-177. F(ab)2 fragments were produced by digestion of trastuzumab IgG (Herceptin) with pepsin for 18 h at 37 °C. The F(ab′)2 fragment fractionated in PD-10 column, followed by the conjugation with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA) as a metal chelator and radiolabeling with 177LuCl3. Molecular weight of fragments was calculated by LCMS (Liquid Chromatography Mass Spectroscopy) and the radiochemical purity was evaluated by ITLC-SG (Instan Thin Layer Chromatography). Our study showed that the purity of F(ab′)2 fragment generated by PD-10 fractions was >98% and the molecular weight of F(ab′)2 was 98.35 kDa. The average numbers of pSCN-Bn-DOTA chelates per antibody fragment were 5.03 ± 1.5 and the optimum conjugation reactions was performed at molar ratio 20:1 (chelator to antibody). The stability test of the radioimmunoconjugate in the human serum albumin (HSA) at 37 °C showed the radiochemical purity was 91.96 ± 0.26% after 96 h storage. This indicated that the radioimmunoconjugate is relatively stable when applied to the human body's physiological condition.