The increase in environmental issues due to the expansion of mass tourism has raised the important of the sustainable development in ecotourism in Vietnam. Plenty of researches emphasized the role of Ecotourism and its impact to the sustainability of tourism businesses. Thereby, this study aims to evaluate factors affecting the development of Ecotourism in Phong Dien district, Can Tho city through the provision of theoretical peer-reviewed studies and this primary research of the author. The results of the study have provided an ample support for a positive relationship between 6 factors to the development of Ecotourism in Phong Dien district, Can Tho city. Particularly, there are six hypotheses being constructed based on the initial academic works. Accordingly, a survey was conducted to examine the expectations and requirements of 150 valid respondents on how to develop ecotourism services. The analysis of Pearson Correlation and Multiple Regression were implemented for the hypotheses testing. The result shows that all six hypotheses have a strongly positive relationship with Ecotourism. Finally, suggestion will be proposed to develop sustainable development for Ecotourism in Phong Dien District. Cantho City.