The invention discloses a gel microsphere used for blood type detection, and the microsphere is connected with a blood type antibody. The invention also discloses a preparation method of the gel microsphere used for blood type detection. The preparation method comprises the following steps: 1) activating groups located on the surface of the gel microsphere, so that the groups can produce chemicalreaction with the blood type antibody to be in joint with the blood type antibody; 2) enabling the blood type antibody to be in joint with the activated groups; and 3) sealing groups which are activated and are not in joint with the blood type antibody by using a sealing agent, and washing, thus gel which can be specifically combined with a blood type antigen is obtained. Compared with an existingblood type detection card, the blood type detection card disclosed by the invention has higher stability and sensitivity as well as a good application prospect.