Accurate and sensitive quantification of testosterone using simple, high-throughput, and low-cost methods has remained a longstanding analytical challenge, despite the issue being highlighted by the endocrinology community more than a decade ago. Existing competitive immunoassays lack sensitivity and specificity, while reference methods based on mass spectrometry are complex, costly, and unsuitable for routine large-scale testing. Alternative approaches are needed to aid in the diagnosis of androgen deficiency and excess in men, women, and children. In this study, we report a novel anti-immunocomplex (anti-IC) antibody that recognizes a monoclonal anti-testosterone antibody bound to testosterone. The anti-IC antibody was generated using phage display, and the antibody pair was utilized in the development of a non-competitive time-resolved fluorescence immunoassay for the detection of testosterone. The assay demonstrated reliable detection within the physiological range of testosterone in men, women, and children and compatibility with plasma as a sample matrix. These findings emphasize the potential of novel anti-IC antibodies in the development of more sensitive immunoassays, offering accessible alternatives to existing methods for testosterone analysis.