Point-of-care testing (POCT) can be performed near the site of the patient to achieve results in a few minutes. Different POCT devices are available in the market, such as microfluidic chips and paper-based lateral flow assays (LFAs). The paper-based LFAs have certain advantages, such as being cheap and disposable, able to detect a wide range of biomolecules, and the fluid flows through them via capillary action eliminating the need for external forces. The LFAs can be optimized for the sensitive and rapid detection of biomolecules. In this study, paper-based fluorescent LFAs platforms using aptamers as the biorecognition molecules were developed for the POCT of insulin. Various parameters were optimized such as concentrations of aptamers, the type of reporter molecules, the volume of sample, and the assay time to quantify insulin levels using a standard LFA reader. The fluorescent LFAs exhibited a linear detection range of 0.1-4 ng.mL-1 with a limit of detection (LOD) 0.1 ng.mL-1. The developed LFAs will help to achieve insulin measurement in a few minutes and will be easy to perform by end-users without the requirement of sophisticated instruments, laboratory set-up, and trained personnel. The developed device will be useful for the measurement of insulin levels in biological samples without the need for pretreatment, reducing the overall cost and time of testing. Moreover, the POCT device were fabricated using paper which is a low-cost (approximately AUD 2 per strip) option and is disposable.Clinical Relevance- POCT monitoring of insulin can facilitate both disease diagnosis and management. The developed LFAs have the capability of rapidly testing insulin concentration within several minutes. It will benefit both patients for at-home daily insulin monitoring and clinicians for hospital rapid insulin testing.