The present disclosure provide a detection chip having at least one functional region, the functional region including: a reaction region and a non-reaction region surrounding the reaction region. The detection chip includes: a first substrate and a second substrate opposite to each other. In the reaction region, a side of the first substrate facing the second substrate is provided with reaction grooves arranged in an array along a first direction and a second direction; first communication grooves each between two reaction grooves adjacent in the first direction, and connected with the two reaction grooves; and second communication grooves each between two first communication grooves adjacent in the second direction, and connected with the two adjacent first communication grooves, and the first direction intersects the second direction. The embodiments of the present disclosure further provide a fabrication method and a sample introduction method of the detection chip.