Survivin is a strong inhibitor of apoptosis protein and a promising target for cancer prevention and treatment. Here， we report the design and preparation of novel chimeric nanobodies (Nbs) that could specifically bind to survivin. We screened the peptides from phage-displayed libraries (7-mer， 12-mer) for nonconserved sequences of complementarity-determining regions (CDRs) in the scaffold of the Nb. By a combination of the nonconserved sequences for CDRs， the corresponding chimeric Nbs (10 Nbs) were prepared with genetic operations. The antisurvivin Nb TAT-Nb4A (a fusion with cellular transduction peptide TAT) was found to be the most efficient antibody on the basis of the results from enzyme-linked immunosorbent assay， MTT， and flow cytometry when these nanobodies were tested with hepatoma carcinoma cell HepG2. TAT-Nb4A could inhibit the growth of HepG2 and promote cancer cell apoptosis significantly in a dose-dependent and time-dependent manner: the apoptosis rate reached 52.5% when the concentration of TAT-Nb4A was 120 μg/ml. Western blotting with cells expressing survivin showed that the prepared nanobody could efficiently bind to expressed survivin and blocked the signaling pathway in which survivin played a role. This study provided a convenient and feasible method of obtaining a novel specific Nb with the case of survivin as a good example.