The development of an in vitro three-dimensional (3D) culture system with cryopreserved biospecimens could accelerate experimental research screening anticancer drugs, potentially reducing costs and time bench-to-beside. However, minimal research has explored the application of 3D bioprinting-based in vitro cancer models to cryopreserved biospecimens derived from patients with advanced melanoma. We investigated whether 3D-printed collagen scaffolds enable the propagation and maintenance of patient-derived melanoma explants (PDMEs). 3D-printed collagen scaffolds were fabricated with a 3DX bioprinter. After thawing, fragments from cryopreserved PDMEs (approximately 1–2 mm) were seeded onto the 3D-printed collagen scaffolds, and incubated for 7 to 21 days. The survival rate was determined with MTT and live and dead assays. Western blot analysis and immunohistochemistry staining was used to express the function of cryopreserved PDMEs. The results show that 3D-printed collagen scaffolds could improve the maintenance and survival rate of cryopreserved PDME more than 2D culture. MITF, Mel A, and S100 are well-known melanoma biomarkers. In agreement with these observations, 3D-printed collagen scaffolds retained the expression of melanoma biomarkers in cryopreserved PDME for 21 days. Our findings provide insight into the application of 3D-printed collagen scaffolds for closely mimicking the 3D architecture of melanoma and its microenvironment using cryopreserved biospecimens.