Accurately predicting durian maturity is a critically unresolved worldwide issue. Farmers currently determine durian ripeness based on their own observation and experience leading to inconsistencies in harvest timing. This reliance on human judgment often results in premature or overripe harvests, impacting fruit quality, yield, and market value. Existing technological solutions, such as sensors are often complex and require specialized expertise, hindering their adoption by farmers and consumers. Developing sensors that can accurately measure durian ripeness without damaging the fruit, are easy to use, and affordable remains a challenge. We introduce a microfluidic paper-based analytical device (μPAD) for on-site, safe matching to meet the demands of durian maturity evaluation. The μPAD automatically collected peduncle fluid without destroying the durian fruit for dual detection of total sugar and amino acid. For determining total sugar including sucrose, glucose, and fructose, several enzymatic steps were reduced to a single step of invertase for sucrose hydrolysis before total reducing sugar was measured using gold nanoparticle (AuNP) generation. Kinetics study of invertase on the μPAD showed Vmax and Km values of 1.42 mM min−1 and 2.17 mM, respectively, that agreed with the direct study of sucrose conversion. To increase device reliability, amino acid was also simultaneously measured with sugar using the simple ninhydrin test with the addition of SnCl2. The developed sensor provided LODs of 3.50, 3.10, 3.30 μM, and 0.02 mg mL−1 for glucose, fructose, sucrose, and amino acid respectively. The μPADs were able to nondestructively discriminate between the mature and immature durians, showing high linear correlation with the standard dry weight method. The development of this μPAD technology has the potential to revolutionize durian cultivation practices, reduce post-harvest losses, and enhance the overall sustainability and profitability of the durian value chain, and can be further developed for maturity tests of other fruits.