B-cell lymphoma remains one of the most refractory tumors, and as such the development of novel treatment approaches, such as antibody-drug conjugates (ADCs), is required. To improve the stability and homogeneity of the ADCs, a humanized anti-CD19 monoclonal antibody (RC58) was developed in the present study. RC58 was based on the CD19 antigen as a potential molecular target of human B-cell lymphomas. RC58 has high CD19-binding affinity and can be internalized in CD19-positive cells through endocytosis. Furthermore, three types of RC58-based ADCs (ADC-1, ADC-2, and ADC-3) were generated using three kinds of Maleimide-PEG-based linkers with two different cytotoxins. The anti-tumor activities of the ADCs were confirmed by in vitro and in vivo experiments. The stability of the ADCs was also evaluated by incubation in human plasma for 10 days. In vitro experiments showed that the three ADCs had distinct inhibitory effects on several B-lymphoma cell lines. Meanwhile, a close correlation between efficacy and drug concentration was found in a nude mouse xenograft model of human B-cell lymphoma, after treatment with RC58-based ADCs. Our results suggest that ADC-1, with high efficiency, could be used as a potential therapeutic agent for human B-cell malignancies.