On-chip cryopreservation of biological cells under low-temperature environments has been successfully demonstrated using a microfabricated chip with an incubation microchamber and microfluidic channels. Microheaters are used as both the resistive heating elements and temperature sensors to control the temperature profile of microenvironment under the liquid nitrogen cooling process. A two-step, temperature-controlled, on-chip cryopreservation process is applied for yeast cells, and after the thawing process, 74% cell survival rate has been accomplished. The reference experiment conducted without using the temperature control results in 27% cell survival rate. As such, this technique could have potential applications in on-chip cryopreservation processes, including those for sperm, embryo, or future cryogenic-based, lab-on-a-chip applications.