Abstract. The natural isoquinoline alkaloid berberine has been demonstrated for its significant activity against a variety of deceases caused by bacterial and parasite infections. Nanodiamond (ND) has also been proven to have great potential for drug delivery. In this work, the complex of ND with berberine is constructed through surface functionalized ND, aiming for drug delivery applications. Spectroscopic analysis was performed on the complex interaction with human lung alveolar carcinoma epithelial cell (A549) and red blood cell (RBC) in comparison with berberine in solution. The studied cells were treated with carboxylated ND, ND-berberine complex and berberine, respectively. Their distributions in cells structures are visualized using ND and berberine's fluorescence signals for detection. The distribution of ND and ND-berberine complex differs with the distribution of berberine introduced in solution form. The results show that the ND with attached drug molecules can act as a drug carrier allowing controllable localization of the drug into the cell. This result can have implication in drug delivery, release and delivery tracing applications. © 2017 Journal of Biomedical Photonics & Engineering.Keywords: Nanodiamond; Berberine; Bio-Applications; Medical Applications; Fluorescence; Surface Functionality; Imaging; Drug delivery; Nanobiotechnology. targeted nanoparticles as specific Helicobacter pylori eradication therapy: in vitro and in vivo study," Nanomedicine (Lond) 10(1), 57-71 (2015). 48. P. R. Vuddanda, A. Mishra, S. K. Singh, and S. Singh, "Development of polymeric nanoparticles with highly entrapped herbal hydrophilic drug using nanoprecipitation technique: an approach of quality by design," Pharm. Dev. Technol.