Wound care management is incredibly challenging for chronic injuries, despite the availability of various types of wound care products in the market. However, most current wound-healing products do not attempt to mimic the extracellular matrix (ECM) and simply provide a barrier function or wound covering. Collagen is a natural polymer that involves a major constituent of the ECM protein, thus making it attractive to be used in skin tissue regeneration during wound healing. This study aimed to validate the biological safety assessments of ovine tendon collagen type-I (OTC-I) in the accredited laboratory under ISO and GLP settings. It is important to ensure that the biomatrix will not stimulate the immune system to produce any adverse reaction. Therefore, we successfully extracted collagen type-I from the ovine tendon (OTC- I) using a method of low-concentration acetic acid. The three-dimensional (3D) skin patch of spongy OTC-I was a soft and white colour, being tested for safety and biocompatibility evaluations based on ISO 10993-5, ISO 10993-10, ISO 10993-11, ISO 10993-23, USP 40 <151>, and OECD 471. For the dermal sensitisation and acute irritation test, none of the tested animals displayed any erythema or oedema effects (p> 0.005). In addition, there were no abnormalities detected in the organ of the mice after being exposed to OTC-I; additionally, no morbidity and mortality were observed in the acute systemic test under the guideline of ISO 10993-11:2017. The grade 0 (non-reactive) based on ISO 10993-5:2009 was graded for the OTC-I at 100% concentration and the mean number of the revertant colonies did not exceed 2-fold of the 0.9%w/vsodium chloride compared to the tester strains ofS. typhimurium(TA100, TA1535, TA98, TA1537), andE. coli(WP2trp uvrA). Our study revealed that OTC-I biomatrix does not present any adverse effects or abnormalities in the present study’s condition of induced skin sensitization effect, mutagenic and cytotoxic towards cells and animals. This biocompatibility assessment demonstrated a good agreement between in vitro and in vivo results regarding the absence of skin irritation and sensitization potential. Therefore, OTC-I biomatrix is a potential medical device candidate for future clinical trials focusing on wound care management.