Antibodies have long been recognized as clinically relevant biomarkers of disease. The onset of a disease often stimulates antibody production in low quantities, making it crucial to develop sensitive, specific, and easy-to-use antibody assay platforms. Antibodies are also extensively used as probes in bioassays, and there is a need for simpler methods to evaluate specialized probes, such as antibody-oligonucleotide (AbO) conjugates. Previously, we demonstrated that thermofluorimetric analysis (TFA) of analyte-driven DNA assembly can be leveraged to detect protein biomarkers using AbO probes. A key advantage of this technique is its ability to circumvent autofluorescence arising from biological samples, which otherwise hampers homogeneous assays. The analysis of differential DNA melt curves (dF/dT) successfully distinguishes the signal from the background and interferences. Expanding the applicability of TFA further, herein we demonstrate a unique proximity based TFA assay for antibody quantification that is functional in 90% human plasma. We show that the conformational flexibility of the DNA-based proximity probes is critically important for optimal performance in these assays. To promote stable, proximity-induced hybridization of the short DNA strands, substitution of poly(ethylene glycol) (PEG) spacers in place of ssDNA segments led to improved conformational flexibility and sensor performance. Finally, by applying these flexible spacers to study AbO conjugates directly, we validate this modified TFA approach as a novel tool to elucidate the probe valency, clearly distinguishing between monovalent and multivalent AbOs and reducing the reagent amounts by 12-fold.