Simple Summary: Royal jelly (RJ) is a nutritious substance secreted by the hypopharyngeal glands (HPGs) of bees, and its nutritional composition may be influenced by feeding factors. There is controversy regarding the relationship between sucrose feeding and the quality of royal jelly. Comparisons were made between RJ obtained from sucrose-fed colonies and honey-fed groups. The results showed that sucrose-fed colonies had higher levels of certain amino acids in the RJ, but no significant differences were found in terms of moisture, protein, glucose, minerals, or other amino acids. Sucrose feeding did not affect the activity of sucrase or the development of the HPGs in nurse bees. Stored food samples from sucrose-fed colonies had higher sucrose levels compared to sealed combs and natural honey. Natural honey had different levels of moisture, Ca, Zn, and Cu compared to honey from sucrose-fed colonies. Overall, sucrose feeding had a minimal impact on the major components of RJ. This study provides important parameter information for further understanding the impact of sucrose feeding on the quality of RJ. Royal jelly (RJ) is a highly nutritious secretion of the honeybees' hypopharyngeal glands (HPGs). During RJ production, colonies are occasionally subjected to manual interventions, such as sucrose feeding for energy supplementation. This study aimed to assess the impact of sucrose feeding on the composition of RJ. The results indicated that RJ obtained from sucrose-fed colonies exhibited significantly higher levels of fructose, alanine, glycine, tyrosine, valine, and isoleucine compared to the honey-fed group. However, no significant differences were observed in terms of moisture content, crude protein, 10-HDA, glucose, sucrose, minerals, or other amino acids within the RJ samples. Moreover, sucrose feeding did not have a significant effect on midgut sucrase activity, HPGs development, or the expression levels of MRJP1 and MRJP3 in nurse bees. Unsealed stored food samples from sucrose-fed bee colonies demonstrated significantly higher sucrose levels compared to sealed combs and natural honey. Additionally, natural honey exhibited higher moisture and Ca levels, as well as lower levels of Zn and Cu, in comparison to honey collected from bee colonies fed sucrose solutions. Based on these findings, we conclude that sucrose feeding has only a minor impact on the major components of RJ.