Zebrafish are a preferred vertebrate model for evaluating metabolism during development, and for toxicity studies. However, commercially available intermittent-flow respirometry systems (IFRS) do not provide a suitable zebrafish-scaled swimming tunnel with a low water volume and proper flow velocities. We developed a miniature IFRS (mIFRS) with a 3D-printed, palm-sized zebrafish treadmill for measuring the swimming ability and metabolic rate of a single one- or three-month-old zebrafish with and without toxicity treatment. The 3D-printed zebrafish treadmill consists of discrete components assembled together which enables the provision of a temporary closed circulating water flow. The results showed that three-month-old zebrafish of normal physiological status had higher energetic efficiency and could swim at a higher critical swimming speed (Ucrit) of 16.79 cm/s with a lower cost of transport (COTopt) of 0.11 μmol g−1m−1. However, for a single three-month-old zebrafish treated with an antibacterial agent, Ucrit decreased to 45% of normal zebrafish and the COTopt increased to 0.24 μmol g−1m−1, due to the impairment of mitochondria. Our mIFRS provides a low-cost, portable, and readily adaptable tool for studying the swimming performance and energetic metabolism of zebrafish.