Natural tissues and organs have different requirements regarding the mechanical characteristics of response. It is still a challenge to achieve biomaterials with anisotropic mechanical properties using an extracellular matrix with biological activity. We have improved the ductility and modulus of the gelatin matrix using 3D printed gelatin microfibers with different concentrations and topologies and, at the same, time achieved anisotropic mechanical properties. We successfully printed flat microfibers using partially cross-linked gelatin. We modified the 10% (w/v) gelatin matrix with microfibers consisting of a gelatin concentration of 14% (w/v), increasing the modulus to about three times and the elongation at break by 39% in parallel with the fiber direction. At the same time, it is found that the microfiber topology can effectively change the matrix ductility, and changing the modulus of the gelatin used in the microfiber can effectively change the matrix modulus. These findings provide a simple method for obtaining active biological materials that are closer to a physiological environment.