The effect of culture in KLD-12 self-assembling peptide nanofiber scaffold containing TGF-beta 3 gene on differentiation of precartilaginous stem cells (PSCs) into chondrocytes was studied. KLD-12 was synthesized by solid-state method. After TGF-beta 3 plasmid was loaded into KLD-12 self-assembling peptide nanofiber scaffold, DNA release ability was investigated. PSCs and hTGF-beta 3 gene were loaded into KLD-12 3-D scaffold, and MTT assay was performed to investigate the cell proliferation, and ELASA assay was used to investigate the expression of TGF-beta 3. Specific cartilage matrix was examined by quantitative real-time PCR, immunohistochemistry and Alcian Blue staining. Compared with control group, DNA synthesis level of PSCs reached the peak within 3 days when PSCs were cultured in self-assembling peptide nanofiber scaffold loading TGF-beta 3 plasmid, and maintained this high level within 2 weeks. MTT results showed that the proliferation ability of experimental group was statistically higher than that in control group (P<0.05). Quantitative real-time PCR suggested that the percentage of TGF-beta 3 positive PSCs in experimental group was higher than that in control group (P<0.01). ELISA assay showed that the TGF-beta 3 protein level increased in supernatant of experimental group's PSCs, reached the peak after 72 h and then declined a little to the plateau phase. Compared with the control group, the specific gene of chondrocyte typical extracellular matrix significantly up-regulated (P<0.01). The results showed that PSCs differentiated into chondrocytes in self-assembling peptide nanofiber scaffold loading TGF-beta 3 plasmid, which provided a fresh approach to cartilage tissue engineering.