Enzyme-Linked Immunosorbent Assay (ELISA) is a versatile and widely used technique to detect and quantify small molecules in various clinical and analytical applications. It is also an effective tool for demonstrating the specificity between antibody and antigen to students across diverse disciplines. However, undergraduate laboratories often face the difficulty of performing proper ELISA exercise due to time and facility constraints. Here, we report a cost-effective, technically simple ELISA exercise which can be completed within 2.5-h at room temperature. This microtitre plate-based ELISA exercise utilises easily sourced inexpensive materials and simulated serum samples which are safe to handle in the undergraduate lab settings. The hands-on procedure and a real-life scenario of disease (Rheumatoid Arthritis) diagnosis reinforce students' understanding of antigen-antibody interactions. ELISA results are displayed by a colourimetric enzymatic reaction, which can be analysed by eye without specialised equipment. This laboratory exercise was designed as part of the final year's immunology course in a three-year undergraduate Biomedical Science programme, but can also be adapted to suit a broad range of student skill levels in science-fields because of its easy set-up and low technical requirement. The procedure and scenario can be also adjusted to meet the specific needs of other educator's goals.