A novel method was developed to prepare an ExonucleaseⅢ-partially-digesting RNA as a competitive reference standard (RNA-CRS) of human stem cell factor (hSCF) gene in quantitative RT-PCR: complete hSCF cDNA was already amplified from HepG2 cells using RT-PCR and cloned into pGEM-T vector. After the recombinant pGEMSCF was treated with Exonuclease Ⅲ and S1 nuclease at a favorable condition to make a limited deletion in hSCF cDNA, the recombinant pGEMSCF mimic was constructed successfully and transcribed in vitro to obtain the RNA-CRS. The hSCF RNA-CRS with a 110 bp deletion from base 499 to 608 in hSCF cDNA was identified by DNA sequencing and it is suitable to be used as a reliable RNA-CRS for the quantitation of the transcriptional expression level of recombinant hSCF in eukaryotic cells by quantitative RT-PCR.