Herein, we present a significant advancement in long-term cryopreservation techniques for microalgae Stichococcus species using a combination of linear polyglycerol (linPG) and dimethyl sulfoxide (DMSO). The technique was tested on three Stichococcus species: Stichococcus bacillaris, Stichococcus deasonii, and Stichococcus minor, which showed long-term viability and recovery rates superior to those when treated with a traditional cryoprotectant only. While DMSO alone enabled high cell recovery rates for all species after 1 week of cryopreservation, the rates for some of them dropped below 50% after 26 weeks of cryopreservation. Treating the cells with a combination of linPG and DMSO raised the recovery rates for all three Stichococcus species to above 92% after long-term cryopreservation. Our findings indicate that linPG in combination with DMSO offers a synergistic and effective solution for maintaining cell integrity and functionality during long-term cryopreservation of microalgae.