Abstract: Midknight Valencia Orange is one of the most important late maturing citrus varieties. In order to study the effect of different harvest maturity on the nutritional quality of this variety and to determine its optimal harvesting period, the quality indexes of Midknight Valencia Orange, including color difference, firmness, carotenoid, soluble sugars, organic acids, free amino acids, mineral elements, flavonoid and total phenol content were measured and a correlation analysis was performed. The result showed that different harvest maturity had significant effects on the quality of Midknight Valencia Orange. With the delay of harvesting time, the coloration process of the peel gradually changed from the stem end to the bottom end and the peel showed a bright yellow after 360 days of flowering. The contents of carotenoid, ascorbic acid, total phenol and flavonoid increased, while the content of soluble solid increased first and then decreased, and the titrable acid content decreased significantly. The main soluble sugars in Midknight Valencia Orange fruit were glucose, sucrose, fructose, talose and inositol, and the contents of glucose and sucrose showed an increasing trend during harvesting. The main organic acids were citric acid, malic acid and quinic acid, and the content of citric acid decreased during harvesting. The main free amino acids were proline, arginine and serine, which showed an increasing trend during harvesting. Based on the evaluation indexes prescribed above, 360~400 days (April~May) after flowering is the appropriate harvesting time for Midknight Valencia Orange. Besides, the correlation analysis results showed that, as the indicator often used to judge the maturity of fruit, the firmness and titratable acid content of Midknight Valencia Orange have highly correlation with the content of carotenoid, soluble sugars, organic acids, free amino acids, mineral elements, flavonoid and total phenol. The firmness, titratable acid content and TSSC/TA ratio of Midknight Valencia can be used for the determination of harvesting time, namely fruit with a firmness 1.33~1.03 kg, titratable acid content 1.77%~1.30% and 5.41~6.96 TSS/TA ratio showed a good quality. The results of this study can provide a scientific basis for the determination the suitable harvesting period of Midknight Valencia Orange.