The invention discloses a reverse transcription primer combination for an efficient reverse transcription reaction. The reverse transcription primer combination is composed of a primer with an oligo(dT) structure and a semi-random primer. The invention further discloses application of the reverse transcription primer combination in the reverse transcription reaction. According to the reverse transcription primer combination, base mismatch and nonrandom pairing which are caused by a common random primer are avoided while the advantages of an anchor primer are retained, the synthesis efficiency preference of a traditional reverse transcription primer to the 5' terminal and the 3' terminal of a mRNA template is eliminated, all luci of the mRNA template can be uniformly synthesized into corresponding cDNA without errors, and then the reverse transcription efficiency and the data uniformity and stability are significantly improved.