Homogeneous assays are advantageous because of their simplicity and rapid kinetics but typically their performance is severely compromised compared to heterogeneous assay formats. Here, we report a homogeneous immunoassay utilizing switchable lanthanide luminescence for detection and site-specifically labeled recombinant antibody fragments as binders to improve the assay performance. Switchable lanthanide luminescence enabled elimination of assay background due to division of the luminescent lanthanide chelate into two non-luminescent label moieties. Simultaneous biomolecular recognition of model analyte cardiac troponin I by two antibody fragments brought the label moieties together and resulted in self-assembly of luminescent mixed chelate complex. The assay was very rapid as maximal signal-to-background ratios were achieved already after 6 min of incubation. Additionally, the limit of detection was 0.38 ng/mL (16 pM), which was comparable to the limit of detection for the heterogeneous reference assay based on the same binders (0.26 ng/mL or 11 pM). This is the first study to apply switchable lanthanide luminescence in immunoassays and demonstrates the versatile potential of the technology for rapid and sensitive homogeneous assays.