BACKGROUND Cardiovascular diseases are the leading cause of death in many countries. Advances in technology have been promoted in this regard, especially in tissue engineering, to meet the need for tissue or organ grafts. In this way, the porcine model has been used due to its morphophysiological similarity between the human species, mainly regarding the cardiovascular system. Tissue engineering is employed using biological scaffolds that are currently derived from porcine. These scaffolds are produced by decellularization, a process to remove cells aiming to maintain only its three-dimensional structure, formed by extracellular matrix (ECM). Its main objective is to produce organs through recellularized scaffolds that could eventually substitute the ones with impaired functions. OBJECTIVE In this way, the present study aimed to establish a new protocol for porcine heart decellularization with potential application on tissue engineering. METHODS A porcine heart aorta was cannulated with a silicon tube, and the organ was washed in 0.1% phosphate-buffered saline through a peristaltic pump (Harvard Peristaltic Pump - Harvard Apparatus). After that, deionized water was introduced in the same system. The decellularization procedure was carried out using ionic and non-ionic detergents, namely 4% sodium dodecyl sulfate (SDS) and 1% Triton X-100, respectively. SDS was perfused through myocardial circulation at 400 mL/min for 24 h for 6 days. Subsequently, the heart was infused with Triton X-100 and washed by PBS and water for 24 h. The heart volume was measured before and after the recellularization. After macroscopic evaluation, the heart samples were processed and stained by Hematoxylin and Eosin, Masson's Trichrome, Weigert-Van Gieson, Alcian Blue, and Pricrosirius Red techniques for microscopic analysis. To observe the cell adhesion, the recellularization was provided in this scaffold, which was analyzed under immunofluorescence and scanning electronic microscopy. RESULTS The protocol provided cells remotion, with adequate concentration of remaining DNA. ECM components as collagen type I, elastin, and glycosaminoglycans were successfully maintained. The scaffold showed a high cells adherence and proliferation in the recellularization process. CONCLUSIONS According to results, the protocol described in this work preserved the ECM components and the organ architecture, minimizing ECM loss and being possible to state that it is a promising approach to tissue bioengineering. This study provides a protocol for whole porcine heart decellularization, which will ultimately contribute to heart bioengineering and may support further studies on biocompatibility relationship of new cells with recellularized scaffolds.