Smart City has become one of the most popular topics in recent years due to the emergence of innovative digital technologies. Start-ups have been active in an innovation system defined as Smart City Entrepreneurial Ecosystem (SCEE) in this study, where stakeholders in the Smart City industry are involved and interact with each other, including start-ups, government, industry players, knowledge institutions, citizens, etc. It is observed that start-ups have encountered the “valley of death” problem in their early development stage with limited commercial resources such as funding, unstable customers, brandings, etc. Hence, it is imperative to research the SCEE and figure out how to help start-ups address these problems by strengthening favourable interactions with other actors in the ecosystem. Since little research has been done on the development and analysis of SCEE, this study aims to develop a theoretical framework of SCEE with the systematic literature review and apply it to analyse the case of Brainport Smart District (BSD) by using Social Network Analysis. The developed theoretical framework consists of sixteen groups of actors under four main categories (i.e., government, academia, industry, and society) and ten types of interactions. As for the social network results, the social network of SCEE is relatively condensed; its diameter is 3, the average clustering coefficient is 0.622, and the average path length is 1.697. Start-ups/SMEs, the local management team of BSD, users & consumers, and software & hardware infrastructure providers are the key players with high values in terms of degree centrality, betweenness centrality, closeness centrality, and eigenvector centrality. When it comes to interactions, most interactions happened between start-ups and actors in the government and industry. And there is also a self-loop in the social network of the SCEE in BSD, which means that start-ups are interacting with other start-ups in the ecosystem. Identifying key actors will be helpful for policymakers or other actors to make strategic decisions to improve the SCEE and create a better environment for start-ups to thrive and grow. The analysis of interactions provides guidance on how to improve the current interactions between start-ups and other actors and points out what interactions are lacking in the ecosystem. This study not only fills in the knowledge gap in the development and analysis of SCEE and benefits scholars interested in this field but also can be adopted by policymakers and practitioners to improve the whole ecosystem and stimulate entrepreneurship and innovation in smart cities.