This study aimed to investigate immobilized metal-organic framework (MOF) UiO-66 and brown-rot fungus Gloeophyllum trabeum (GT) in PVA-SA matrices for adsorption and decolorization of reactive black 5 (RB5). Furthermore, UiO-66/GT@PVA-SA composite was successfully fabricated and obtained by immobilizing UiO-66 and GT mycelia into a mixture of PVA-SA. This composite demonstrated a decolorization ability of 80.12% for RB5 after 7 days. The composite's reusability was assessed for three cycles; at last, it only achieved 21%. This study reported that adsorption of RB5 by the composite followed a pseudo-second-order kinetic model with a correlation coefficient (R2) of 0.9997. The Freundlich model was found to be suitable for the isotherm adsorption. The process was also spontaneous and feasible, as indicated by the negative ΔG value. Subsequently, four metabolite products resulting from decolorization of RB5 by UiO-66/GT@PVA-SA composite were proposed, namely: C24H19N5Na2O13S4 (m/z = 762), C10H13N2O8S2− (m/z = 353), C12H9N4O7S2− (m/z = 384), and C10H13O8S2− (m/z = 325).