Heparin is a vital macromolecule that regulates blood coagulation, while protamine is an essential polypeptide clinically used to counteract heparin overdose. Detecting both heparin and its antidote protamine under physiological conditions is crucial for biological and clinical applications. This report introduces a cucurbituril[8] (CB[8])-based phosphorescent probe for their detection. The method employs a nanoassembly induced phosphorescence switch-on mechanism for heparin sensing and a disassembly induced phosphorescence switch-off approach for protamine detection. An arginine-rich guest forms a supramolecular complex with heparin, enhancing phosphorescence under secondary confinement and enabling its detection. Conversely, protamine sulfate, as a stronger competitor for heparin, disrupts the probe-heparin aggregates, leading to emission quenching and protamine sensing. This sensor demonstrated high selectivity in detecting both analytes in biological samples, such as human blood serum and urine. The detection limits for heparin and protamine were determined to be 61 and 82 ng/mL in 10% HBS, respectively.