Performing realistic and reliable in vitro biological dose verification with good resolution for a complex treatment plan remains a challenge in particle beam therapy. Here, a new 3D bio-phantom consisting of 96-well plates containing cells embedded into Matrigel matrix was investigated as an alternative tool for biological dose verification. Feasibility tests include cell growth in the Matrigel as well as film dosimetric experiments that rule out the appearance of field inhomogeneities due to the presence of the well plate irregular structure. The response of CHO-K1 cells in Matrigel to radiation was studied by obtaining survival curves following x-ray and monoenergetic 12C ion irradiation, which showed increased radioresistance of 3D cell cultures in Matrigel as compared to a monolayer. Finally, as a proof of concept, a 12C treatment plan was optimized using in-house treatment planning system TRiP98 for uniform cell survival in a rectangular volume and employed to irradiate the 3D phantom. Cell survival distribution in the Matrigel-based phantom was analyzed and compared to cell survival in a reference setup using cell monolayers. Results of both methods were in good agreement and followed the TRiP98 calculation. Therefore, we conclude that this 3D bio-phantom can be a suitable, accurate alternative tool for verifying the biological effect calculated by treatment planning systems, which could be applied to test novel treatment planning approaches involving multiple fields, multiple ion modalities, complex geometries, or unconventional optimization strategies.