As a biomaterial, besides excellent biocompatibility and biodegradability, suitable macropores and pores structure should be provided to guide cell extension and migration. In present study, the silk fibroin (SF) scaffold with uniaxial channels was prepared by directional temperature field freezing technique. The average pore diameter, pore density and porosity of the scaffold with oriented channels are ${sim}128.7{mu}m$, ${sim}158mm^{-2}$ and ~91.4 %, respectively. By controlling of the temperature gradient direction, the oriented multichannels of the scaffolds were formed in longitudinal easily. In process of the scaffolds fabrication, the directional growth of ice crystal could shear and draft to the silk fibroin molecule segments, which resulted in the new crystal nucleus formation in new zone and increase of ${eta}$-sheet components in the scaffolds. In vitro, L929 cells were seeded on the scaffolds with oriented channels to evaluate the effect on cell behavior. Cell viability, adhesion and morphology were determined by methyl thiazolyl tetrazolium, confocal microscope and scanning electron microscope. The results showed that the cells anchored on the oriented channels, spread along the direction of the channels and hold a higher viability on the scaffolds with oriented channels. These new oriented multichannel scaffold could guide the adhesion and proliferation of L929 cells, which hold a potential in tissue engineering.