The g-C3N4/CF composite carrier was prepared by ultrasound-assisted maceration and high-temperature calcination. The enzyme immobilization using the g-C3N4/CF as the novel carrier to immobilize lactase and glucose isomerase was enhanced for lactulose production. The carbon fiber (CF) was mixed with melamine powder in the mass ratio of 1:8. The g-C3N4/CF composite carrier was obtained by calcination at 550 °C for 3 h. After the analysis of characteristics, the g-C3N4/CF was successfully composited with the carbon nitride and CF, displaying the improvement of co-immobilization efficiency with the positive effects on the stability of the enzyme. The immobilization efficiency of the co-immobilized enzyme was 37% by the novel carrier of g-C3N4/CF, with the enzyme activity of 13.89 U g−1 at 60 °C. The relative activities of co-immobilized enzymes maintained much more steadily at the wider pH and higher temperature than those of the free dual enzymes, respectively. In the multi-batches of lactulose production, the relative conversion rates in enzymes co-immobilized by the composite carrier were higher than that of the free enzymes during the first four batches, as well as maintaining about a 90% relative conversation rate after the sixth batch. This study provides a novel method for the application of g-C3N4/CF in the field of immobilizing enzymes for the production of lactulose.