Quality and yield are the primary concerns in kiwifruit breeding, but research on the genetic mechanisms of fruit size, shape, and ascorbic acid (ASA) content is currently very limited, which restricts the development of kiwifruit molecular breeding.  In this study, we obtained a total of 8.88 million highly reliable single nucleotide polymorphism (SNP) markers from 140 individuals from the natural hybrid offspring of Actinidia eriantha cv. ‘White’ using whole genome resequencing technology.  A genome-wide association study was conducted on eight key agronomic traits, including single fruit weight, fruit shape, ASA content, and the number of inflorescences per branch.  A total of 59 genetic loci containing potential functional genes were located, and candidate genes related to single fruit weight, fruit length, ASA content, number of inflorescences per branch and other traits were identified within the candidate interval, such as AeWUSCHEL, AeCDK1 (cell cycle dependent kinase), AeAO1 (ascorbic oxidase) and AeCO1 (CONSTANS-like 4).  After constructing an RNAi vector for AeAO1 and injecting it into the fruit of cv. ‘Midao 31’ to interfere with the expression of the AeAO1 gene, the results showed that the activity of ascorbic oxidase in the fruit of ‘Midao 31’ significantly decreased, while the content of ASA significantly increased.  This study provides valuable insights into the genetic basis of variation in A. eriantha fruit traits, which may benefit molecular marker-assisted breeding efforts.