Gold nanocluster (AuNC) synthesis using a well‐distinguished polymer for nanoparticle‐mediated drug delivery paves the way for developing efficient theranostics based on pharmaceutically accepted materials. Gelatin‐stabilized AuNCs are synthesized and modified by glutathione for tuning the emission spectra. Addition of silver ions enhances the fluorescence, reaching also high quantum yield (26.7%). A simplified model can be proposed describing the nanoclusters' properties–structure relationship based on X‐ray photoelectron spectroscopy data and synthesis sequence. Furthermore, these modifications improve fluorescence stability toward pH changes and enzymatic degradation, offering different AuNCs for various applications. The impact of nanocluster formation on gelatin structure integrity is investigated by Fourier transform infrared spectrometry and matrix‐assisted laser desorption/ionization time of flight mass spectroscopy, being important to further formulate gelatin nanoparticles (GNPs). The 218 nm‐sized NPs show no cytotoxicity up to 600 µg mL−1 and are imaged in skin, as a challenging autofluorescent tissue, by confocal microscopy, when transcutaneously delivered using dissolving microneedles. ... mehrLinear unmixing allows simultaneous imaging of AuNCs–GNPs and skin with accurate signal separation. This underlines the great potential for bioimaging of this system to better understand nanomaterials' behavior in tissue. Additionally, it is drug delivery system also potentially serving as a theranostic system.