Dermatophytosis is a widely spread contagious zoonotic disease, affecting both man (tinea) and animals (ringworm). This disease is caused by a group of closely related keratinophilic fungi known collectively as the dermatophytes group. Although the wide distribution of dermatophytosis cases throughout the whole world and its adverse clinical effect on human health, economical effect on productive animals, and pet animal welfare, there is no rapid accurate diagnostic tool for such disease. The current conducted study tries to accomplish the difficult equation by achieving an accurate, sensitive, specific, user-friendly, rapid, robust, device-less, deliverable to end-users, and economic cost for the development and production of diagnostic kits. Through the development of a rapid diagnostic kit based on immunochromatographic assay with three major affordable reproducible production stages; preliminary stage, developmental and standardization stage, and evaluation stage. Obtaining dermatophytes-specific polyclonal antibodies against criteria-based selected dermatophytes strains associating proper gold nanoparticle preparation, characterization, and conjugation, with proper loading of the different bio-reactants on the efficiently laminated and fabricated lateral flow strips were the main challenge and control points through the whole process. Also, as a result of examining 100 animal samples using the new kit, the κ coefficients of the kit with the direct microscopy while the kit with the culture were 0.44 and 0.76, respectively. Therefore, the newly designated and developed kit showed a very promising competitive diagnostic result within 5–7 min through easy-to-be-performed three steps.