The aim of this study was to determine an efficient whole-organ decellularisation protocol of a human-sized testis by perfusion through the testicular arteries. In the first step of this study, we determined the most efficient detergent agent, whereas the second phase delineated the optimal time required for the decellularisation process. Initially sheep testes were decellularised by one of three different detergent agents: sodium dodecyl sulphate (SDS), Triton X-100 and trypsin-ethylenediamine tetraacetic acid (EDTA) solutions, each perfused for 6h. In the second phase, the selected detergent agent was applied for different time periods. A total number of 20 organs were processed during this investigation. The efficacy of the decellularisation process and the preservation of the extracellular matrix components and structure were evaluated by histopathological examinations, 4',6'-diamidino-2-phenylindole (DAPI) staining, DNA quantification, hydroxyproline measurement, magnetic resonance imaging and scanning electron microscopy. Organ perfusion with 1% SDS solution for 6 to 8h demonstrated the most desirable outcomes regarding decellularisation and extracellular matrix preservation. The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay was used to determine the toxicity of the scaffold and its potential for further application in tissue-engineering investigations. This investigation introduces an efficient method to produce a three-dimensional testicular bio-scaffold resembling the properties of the native organ that could be employed in tissue-engineering studies.