Banana in Indonesia is one of the horticultural commodities with high economic value and widely cultivated in all regions. Banana is used not only for fresh food but also for processed products such as flour to reduce the use of wheat flour, which currently Indonesia imports in large quantities. This study aimed to determine the effect of differences in fruit maturity levels of three local Balinese banana cultivars on the quality of the banana flesh flour produced. The study used a randomized block design with 2 factors and 3 replications. The first factor was the local Balinese banana cultivar consisting of 3 levels (Ambon Kuning, Kepok, and Ketip banana), while the second factor was the maturity level of the fruit at harvest which also consists of 3 levels (unripe, physiologically ripe, and overripe). The results showed that the interaction between local Balinese banana cultivars and maturity level of the fruit at harvest was significantly different for all observed variables except for the heavy metal content of lead. Based on the various variables observed, the quality of banana flesh flour in the combination of Balinese banana cultivars and degree of maturity of the fruit at harvest is relatively adequate as a wheat flour substitute. Unripe Ketip banana produce the best quality banana flesh flour, with a weak smell, a powdery form, the color L*81.41, protein content of 5.01%, iron content of 211.64%, and lead content of 0.75%.