The disclosure provides fusion reporter protein constructs and related compositions, systems, and methods for nanopore-based detection biological activity. In one aspect, the disclosure provides a fusion reporter protein comprising, in order: a blocking domain with a stably folded tertiary structure; a flexible analyte domain; and a flexible tail domain, wherein the flexible tail domain has a net negative charge. The disclosure also provides nucleic acid constructs encoding the disclosed fusion reporter protein, and vectors and cells comprising the nucleic acids. Also provided are nanopore-based systems and methods for using the disclosed fusion reporter protein constructs to detect and characterize biological activity.