<p>Accurate diagnosis of myocardial injury is crucial in clinical practice, as it enables prompt treatment, reduces the risk of complications, and improves patient outcomes. Examining cardiac troponin composition offers distinct advantages as it provides valuable insights that help differentiate between ischaemic and non-ischaemic causes of cardiac injury, thereby directly enhancing clinical assay specificity. Current high-sensitivity troponin assays target all circulating forms of either troponin I or T but are unable to distinguish individual forms or specific combinations. This is an issue as troponin is also elevated in non-cardiac conditions, and emerging evidence suggests that the ratio between troponin I and TIC complex isoforms may serve as an indicator for differentiating acute from chronic myocardial injury. Here, we introduce a methodology utilising a label-free Surface Plasmon Resonance biosensor to detect and differentiate between troponin I and complex isoforms. Unlike traditional immunoassay, which requires indirect labelling with secondary antibodies to detect these biomarkers, this method provides a real-time detection of these isoforms, as each antibody interaction with the specific analytes provides a quantitative reading. We demonstrated that this method can detect complexed and free troponin I in varying ratios. This method has the potential to be developed into point-of-care testing and opens avenues for isoform-specific prognostic assays.</p>