There are a number of significant changes taking place in modern city development and most of them are based on the number of recent technological progress. This paper provides a review and analysis of recent approaches of biotechnology that can find a place in today’s cities and discusses how those technologies can be integrated into a city’s Internet of Things (IoT). Firstly, several biotechnologies that focus on rain gardens, urban vertical farming systems, and city photobioreactors are discussed in the context of their integration in a city’s IoT. The next possible application of biofuel cells to the sensor network’s energy supply is discussed. It is shown that such devices can influence the low-power sensor network structure as an additional energy source for transmitters. This paper shows the possibility of bioelectrochemical biosensor applications, discusses self-powered biosensors, and shows that such a system can be widely applied to rainwater monitoring in rain gardens and green streets. Significant attention is paid to recent approaches in synthetic biology. Both cell-based biosensors and bioactuators with synthetic genetic circuits are discussed. The development of cell-based biosensors can significantly enhance the sensing possibilities of a city’s IoT. We show the possible ways to develop cyber-physical systems (CPSs) with the systems mentioned above. Aspects of data handling for the discussed biotechnologies and the methods of intelligent systems, including those that are machine learning-based, applied to the IoT in a city are presented.