Sea cucumbers are vital for the health of ocean ecosystems as they help recycle nutrients, benefiting other marine life. They are considered a delicacy in China and other Asian countries, largely due to their reported medicinal properties. This heightened interest has led to overfishing and the potential extinction of some species. Therefore, there is an urgent need to mitigate the negative impact of fishing on sea cucumber populations through aquaculture. Current aquaculture methods for inducing spawning are inefficient, leading to a low number of juveniles that could replenish ocean beds. This study aimed to use multi-omics tools on Holothuria leucospilota (black sea cucumber) to investigate the key molecular components that regulate reproductive processes in sea cucumbers. With a focus on neural and gonad tissues, RNA was isolated and changes in gene expression were investigated at five reproductive stages that correlated with gonad maturation. In combination with proteomics, we have identified recognised reproduction-related proteins, as well as novel proteins that increase throughout gonad maturation. This includes proteins secreted into the coelomic fluid, which could serve as potential biomarkers for maturation; an accurate and non-invasive approach for determining the maturation stage would greatly benefit the sea cucumber aquaculture industry. In summary, this omics investigation has led to a new understanding of the molecular components associated with advancing maturation and spawning in a sea cucumber, which could lead to the development of diagnostic tools for the maturation stage, as well as hormones that may help overcome reproductive dysfunction.