Human bocaparvovirus 1 (HBoV1) is one of the two parvoviruses that infect humans and cause diseases. Infection with HBoV1 in infants and young children aged 2−5 years can lead to mild or severe acute respiratory diseases, with the most severe cases posing a life-threatening risk. Similar to other parvoviruses, the HBoV1 DNA genome consists of two terminal reverse repeats (ITRs) at its ends, which are necessary for viral genome replication. However, up to now, it has remained a technical challenge to clone the entire ITRs through PCR amplification. In this study, we successfully constructed a full-length infectious clone of HBoV1, termed as pSKHBoV1, by synthesizing and cloning the terminal ITRs in a stepwise manner. After transfecting HEK293 cells with the infectious clone pSKHBoV1, we were able to reconstitute the viral replication cycle. This included the expression of key non-structural proteins, post-transcriptional modification and processing of viral RNA, viral genome replication, and potentially the production of progeny virions containing the defined DNA genome. The successful construction of the infectious clone pSKHBoV1 lays the foundation for future studies on HBoV1 replication and propagation, virus-host interaction, and the development of viral vaccines.