Conventional laboratory protein detection techniques are not suitable for point-of-care (POC) use because they require expensive equipment and laborious protocols, and existing POC assays suffer from long development timescales. Here, we describe a modular cell-free biosensing platform for generalizable protein detection that we call TLISA (T7 RNA polymerase-linked immunosensing assay), designed for extreme flexibility and equipment-free use. TLISA uses a split T7 RNA polymerase fused to affinity domains against a protein. The target antigen drives polymerase reassembly, inducing reporter expression. We characterize the platform and then demonstrate its modularity by using 16 affinity domains against four different antigens with minimal protocol optimization. We show that TLISA is suitable for POC use by sensing human biomarkers in serum and saliva with a colorimetric readout within 1 hour and by demonstrating functionality after lyophilization. Altogether, this technology has the potential to enable truly rapid, reconfigurable, modular, and equipment-free detection of diverse classes of proteins.