Nanoparticle (NP) implementation in industry has increased during the last years. However, the consequences of NP release to the environment have not been fully understood yet and long-term effects are hardly predictable. Information regarding a possible NP uptake by organisms is urgently needed. Here, we investigate the uptake of presynthesized AuNPs of different diameters by living diatoms. To better understand the influence of NPs on diatoms, cells were monitored in vivo using Surface Enhanced Raman Spectroscopy (SERS). By 3D Raman imaging, AuNPs with diameters ≥50 nm could clearly be localized inside the cells, whereas smaller AuNPs were never detected in the cell interior. This indicates a size-dependent uptake mechanism that comes along with different toxicities. As diatoms present an essential source of the marine food chain, this observation is relevant not only for diatoms themselves but also for higher organisms.