The invention discloses a Citrin deficiency gene detection primer, comprising an upstream prim as shown in SEQ ID No. 1, a downstream primer as shown in SEQ ID No. 2 and a probe as shown in SEQ ID No.3. The invention also discloses the use of the detection primer in preparing a Citrin defect detection reagent and a Citrin defect detection reagent kit. The detection primer provided by the invention can realize the simultaneous detection of the wild type of the c.851_854del (rs80338720) of the disease-causing gene SLC25A13 of the Citrin deficiency in the same tube PCR reaction, Heterozygous mutation and homozygous mutation have the advantages of simple operation, high sensitivity, high specificity and low cost, which are very suitable for the detection and diagnosis of clinical Citrin deficiency.