The present invention provides a primer pooi for amplifying a target sequence of a human glucokinase gene including at least one set of primers or variant primers thereof, each set of primers being identified by two consecutive SEQ ID NOS, the SEQ ID NOS starting at SEQ ID NO:3 and terminating at SEQ ID NO:24 according to the present invention. A target sequence such as the human glucokinase gene may be amplified with a high specificity, a high speed, a high sensitivity and a low cost through a multiplex PCR to detect a maturity-onset of diabetes of the young (MODY) 2 associated gene.