Practical applications of high-TC superconducting quantum interference devices (SQUIDs) require cheap, simple in operation, and cryogen-free cooling. Mechanical cryo-coolers are generally not suitable for operation with SQUIDs due to their inherent magnetic and vibrational noise. In this work, we utilized a commercial Joule–Thomson microfluidic two-stage cooling system with base temperature of 75 K. We achieved successful operation of a bicrystal high-TC SQUID gradiometer in shielded magnetic environment. The micro-cooler head contains neither moving nor magnetic parts, and thus does not affect magnetic flux noise of the SQUID even at low frequencies. Our results demonstrate that such a microfluidic cooling system is a promising technology for cooling of high-TC SQUIDs in practical applications such as magnetic bioassays.