Glucoraphanin is an important glucosinolate which is widely distributed in Brassica vegetables and poses an anticancer effect to humans. Although researchers have paid a lot of attention to the changes in glucoraphanin concentration in seedlings of broccoli over 1–2 weeks, there has been little research focusing on the total whole-sprout glucoraphanin content within broccoli seedlings over 1–5 weeks. However, it is necessary to clarify the changes in total glucoraphanin content during the broccoli sprouting stage as broccoli seedlings are novel plant foods. This research explored glucoraphanin absolute accumulation and the biosynthesis mechanism in broccoli seedlings during a 5-week growth period. The results showed that glucoraphanin accumulation content was higher at week 4 than in the seeds. Moreover, the relative DL-methionine contents increased significantly after 3 weeks. Glucoraphanin synthetic gene expression levels were increased after 3 weeks, but the gene expressions of AOP3 (encoding 2-oxoglutarate-dependent dioxygenases) and MYR (encoding myrosinase) were significantly decreased. Furthermore, the 20 essential DEGs obtained can provide new insight into understanding the developmental regulation of broccoli seedlings. In addition, the results can also provide information on how to obtain higher glucoraphanin contents in broccoli sprouts.