The invention discloses a habituation culture method of psychrophilic methanogens. The habituation culture method comprises the following steps: 1) feeding a habituation culture medium into a culture device, introducing nitrogen to remove oxygen, sterilizing at high temperature, and adding 0.001g of sodium acetate, sodium formate, methanol, cellulose acetate, chitosan and prebiotics respectively, thereby obtaining a habituation culture device; 2) crushing sludge bacteria, adding activated starch, and putting the mixture to stand, thereby obtaining an activated starch-sludge bacterium; 3) inoculating the bacterium obtained in the step 2) to the habituation culture device of the step 1), and culturing for 4-5 days at 8 DEG C; 4) detecting, changing the temperature through a temperature control system when bubbles are generated, and implementing temperature-variable habituation culture; and 5) stopping temperature variation when the bubbles are not increased any more, and replenishing materials to generate bubbles continuously until the gas generation amount is completely stable after 12 days, thereby completing enrichment of psychrophilic methanogens. By adopting the habituation culture method of psychrophilic methanogens, the formation speed of bacterial colonies is remarkably increased, and the enrichment effect is improved.