Objective To explore an ideal method for culturing autologous chondrocytes while maintaining their cartilaginous phenotype by combining monolayer culture with three-dimensional culture. Methods Chondrocytes were cultured by routine monolayer culture method, then the chondrocytes of fourth passage were seeded into alginate beads for three-dimensional culture following standard protocols. The cell morphology was observed by inverted microscope at predefined time points. The cell viability and proliferation were tested by trypan blue and LIVE/DEADR ○ Kit, respectively. And extracellular matrix formation was examined by toluidine blue staining. Results The cartilaginous phenotype of chondrocytes could only be maintained by passage 2 and 3 after monolayer culture. Three-dimensional culture could gradually regain and maintain the cartilaginous phenotype of de-differentiated chondrocytes (P4). Cells of P1-3 by monolayer culture could maintain 90% activity, which decreased from P4.The cytoactivity of cells by three-dimensional culture method was always above 90%. The total doubling rate till P6 by monolayer culture (28 days) was 44 times that by three-dimensional culture. The staining intensity of cartilaginous extracellular matrix was significantly decreased after P4 by monolayer culture (P<0.05). For three-dimensional method, the staining intensities of cartilaginous extracellular matrix at day 21 and 28 were significantly higher than that at day 7(P<0.05). Conclusion Monolayer culture allows proper proliferation of chondrocytes, and following three-dimensional culture in alginate beads can regain the cartilaginous phenotype of chondrocytes, thus achieving the aim of both amplification and maintenance of the cartilaginous phenotype.