The development of porous biodegradable scaffolds is of great interest in tissue engineering. In this regard, exploration of novel biocompatible materials is needed. Silk fibroin-chondroitin sulfate-sodium alginate (SF-CHS-SA) porous hybrid scaffolds were successfully prepared via lyophilization method and crosslinked by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-ethanol treatment. According to the scanning electron microscopy studies, mean pore diameters of the scaffolds were in the range of 60–187 μm. The porosity percentage of the scaffold with SF-CHS-SA ratio of 70 : 15 : 15 (w/w/w %) was 92.4 ± 3%. Attenuated total reflectance Fourier transform infrared spectroscopy, X-ray diffraction, and differential scanning calorimetry results confirmed the transition from amorphous random coil to crystalline β-sheet in treated SF-CHS-SA scaffold. Compressive modulus was significantly improved in hybrid scaffold with SF-CHS-SA ratio of 70 : 15 : 15 (3.35 ± 0.15 MPa). Cytotoxicity assay showed that the scaffolds have no toxic effects on chondrocytes. Attachment of chondrocytes was much more improved within the SF-CHS-SA hybrid scaffold. Real-time polymerase chain reaction analyses showed a significant increase in gene expression of collagen type II, aggrecan, and SOX9 and decrease in gene expression of collagen type I for SF-CHS-SA compared with SF scaffold. This novel hybrid scaffold can be a good candidate to be utilized as an efficient scaffold for cartilage tissue engineering. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 41048.