Picobirnavirus (PBV) is a small (35 nm in diameter, i.e., “pico”), non-enveloped, bi-segmented dsRNA (i.e., “bi-RNA”) virus. Since the discovery of PBV in 1988, PBV has been detected in various host species, including humans, mammals, birds, reptiles, and even in environments like sewage. The actual host of PBV remains controversial, and no infection model has been established to unveil the pathology or pathogenesis associated with PBV infection. In this study, I constructed a plasmid-based expression system for hPBV by expressing the two viral RNA segments in the E. coli Rosetta 2 strain. All predicted viral proteins, i.e., ORF1, ORF2, CP, and RdRP, were detected upon the viral RNA expression, indicating efficient translation from inherent ribosomal binding sites. Purified recombinant hPBV capsids were found to comprise: both viral RNA segments, ORF2, and RdRP. Viral RNA segments were preferentially packaged into the capsids over host mRNAs, presumably due to packaging signal (PS) sequences at the terminal ends. Such terminal PS sequences were able to carry a non-viral RNA sequence into the recombinant hPBV. ORF2 protein was observed in the VLPs for the first time. By engineering a series of truncation mutants, it was determined that the N- terminal domain of ORF2 is responsible for its incorporation into the capsids. Through immunoprecipitation assays, it was found that hPBV RdRP directly interacts with CP into assembly intermediates. Still, RdRP was only present at deficient levels in assembled capsids at its overexpression. This work presents a plasmid-based expression system to fully dissect molecular determinants for hPBV assembly and genome packaging. Results from this study indicate that hPBV is indeed a prokaryotic virus, but E. coli is likely not the natural host of hPBV. Co-immunoprecipitation of recombinant hPBV with lysates of different microbiota species in future studies would reveal the identity of the hPBV native host and establish the PBV infection model.