Universities play a strategic role as drivers of regional economic growth. One contribution is the establishment and support of University Spin-off companies (USOs), as a knowledge transfer mechanism, thus contributing to generating entrepreneurial university ecosystems. Recent literature points to this as a promising and emerging research area and policymakers are increasingly investing in universities to facilitate economic growth. This is based partially on the idea that a well-established or successful entrepreneurial university ecosystem automatically leads to the emergence of business ecosystems with subsequent benefits to the region. Using the knowledge spillover theory conceptual lens in combination with a social networks approach, this is explored in the region of Andalusia (Spain), centring on the regional entrepreneurial university ecosystem and in the role of the USOs. By means of a mixed-methods approach, we identified the main actors of the Andalusian entrepreneurial university ecosystem and found how the ecosystem is enacted, using a social network approach, and how the ecosystem promotes an expansive wave effect making the knowledge spillover possible to businesses beyond the entrepreneurial university ecosystem. This study confirms empirically that the USOs are main actors in the entrepreneurial university ecosystem and strengthen knowledge transfer, by relating with other businesses beyond this ecosystem; and expands the emergent ecosystem approach into the field of entrepreneurship.