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Curdlan (β-1, 3-glucan) is a kind of exopoly-saccharide excreted by bacteria and has the ability of improvement of immune and anticancer. It has potential values as medical material in medicine field. Many researchers began to focus on the interaction of curdlan and immune signal protein for finding the detail mechanism of its bioactivity. The aim of this work was to investigate the effect of antibiotics and ammonia additions in combination on the enhancement of curdlan biosynthesis. A two-stage fed-batch process was used for curdlan production in shake flask culture of Alcaligenes faecalis. With the optimized concentration of 100mg/L of ampicillin was added to the culture medium and fermented for 5 days, it was achieved a concentration of 24.65g/L curdlan and a yield of 10.67 curdlan g/g cell, which were 2.02-fold and 2.1-fold of those without addition of ampicillin. With the streptomycin instead of ampicillin, a similar enhancement of curdlan production was observed, reaching a yield of 11.78 g/g cell and a concentration of 20.77g/L curdlan. It was demonstrated that the enhancement of curdlan production was related to the increased activity of key enzymes involving curdlan biosynthesis. At the same time, the cell wall biosynthesis in the bacterium was inhibited by antibiotics.