The invention provides a haemophilus culture medium and a preparation method thereof. The haemophilus culture medium comprises the following components by weight percent: 0.3-3% of Columbia agar, 0.001-0.003% of X factor, 0.001-0.003% of Y factor and 0.0005-0.002% of bacitracin disk antibacterial agent, and the culture medium is prepared by heating and mixing the components; the culture medium is rich in nutrition, the consistency of culture capacity is high, the growth of bacteria is easily observed, by maintaining the normal activity of the factor V and factor X under specific conditions, Haemophilus influenzae grows stably, the culture medium has strong anti-bacterial capability, the haemophilus can be effectively separated, the cost is greatly reduced, the direct cost is equivalent to only about 30% of that of foreign modified chocolate agar; and the preparation method is simple and suitable for the requirement on large-scale industrial production.