Reverse transcription uses the reverse transcriptase enzyme to synthesize deoxyribonucleic acid (DNA) from a ribonucleic acid (RNA) template. This plays an essential role in viral replication. There are still, however, many unknown facts regarding the timing and dynamic processes involved in this life stage. Here, three types of dual-fluorescence human immunodeficiency virus type-1 (HIV-1) particles were constructed with high infectivity, and the sequential process of reverse transcription was observed by real-time imaging of a single HIV-1 particle. Viral uncoating occurred at 60-120 min post infection. Subsequently, at 120-180 min post infection, the viral genome was separated into two parts and reverse-transcribed to generate a DNA product. Nevirapine (NVP), a reverse transcriptase inhibitor, can delay the dynamic process. This study revealed a delicate, sequential, and complex relationship between uncoating and reverse transcription, which may facilitate the development of antiviral drugs.