Fluorescence microscopy is one of the important instruments used in research and clinical laboratories on an everyday basis due to its wide range of applications ranging from a diagnostic tool to a molecular imaging device. However, the high cost, complex instrumentation, and requirement of sophisticated facilities limit its availability to school and college students, especially in developing and underdeveloped countries. In this work, we report the design and development of a slide-like portable total internal reflection fluorescence (TIRF) adapter, which can be integrated easily with Foldscope (a paper microscope) to observe and capture the fluorescence images using any smartphone. The adapter consists of a battery-powered LED (light source), a small piece of multimode optical fiber, a slide-like 3-D printed waveguide platform to hold the optical fiber and light emitting diode (LED) at an appropriate position. The potential of the adaptor is validated by capturing the fluorescence images of two most commonly used dyes i.e. fluorescein isothiocyanate (FITC) and rhodamine-B, after being immobilized on amine-functionalized optical fiber probe. Further, this adaptor is successfully used to demonstrate (i) Förster resonance energy transfer phenomenon by employing FITC dye as donor and gold nanoparticles as quencher moiety and (ii) fluorescence bioassay by observing antigen-antibody interactions using human immunoglobulin G as the receptor and FITC-tagged goat anti-human immunoglobulin G as analyte. The inexpensive, portable and user-friendly TIRF adapter is a potential alternative to conventionally used fluorescence microscopes, especially for the school and college students and in resource-constrained clinical laboratories. © 2019 IEEE.