The tempeh processing process produces waste in the form of liquid and solid waste that can be utilized into eco-enzyme with the addition of yeast and substrate and lemon peel as a companion raw material. This study aims to produce eco-enzyme from tempeh waste which is then applied as a preservative for mango fruit. The fixed variables in this study are the volume of liquid waste, the amount of epidermis solid waste, the amount of lemon peel, and the amount of yeast and the length of fermentation time. The independent variables were fermenter type (1 and 2) and substrate type (brown sugar (Eco-A), granulated sugar (Eco-B), and molasses (Eco-C)). The results showed that the eco-enzyme products produced were light brown (Eco-A and Eco-C), light yellow (Eco-B) and had a fresh sour aroma typical of lemon peel. The volume of eco-enzyme products after fermentation process were Eco-A1 3.45; Eco-A2 3.27; Eco-B1 3.35; Eco-B2 3.55; Eco-C1 3.25; and Eco-C2 3.36 L. The pH value obtained ranged from 3.05-3.25. Phenol levels ranged from 0.050-0.175mg/L. The fruit preservation process obtained with Eco-C1 is the best variable, where the mango fruit has a shelf life of up to 16 days, with commercial organic preservatives for 12 days and mango fruit without preservatives has a shelf life of 10 days. The preservative from tempeh waste is potential to develop as a commercial product.