Gold nanorods (GNRs) can be functionalized with multiple biomolecules allowing efficient cell targeting and delivery into specific cells. However, various issues have to be addressed prior to any clinical applications. They involve controlled biofunctionalization to be able to deliver a known dose of drug by immobilizing a known number of active molecules to GNRs while protecting their surface from degradation. The most widely used synthesis method of GNRs is seedmediated growth. It requires the use of cetyltrimethylammonium bromide (CTAB) that acts as a strong capping agent stabilizing the colloidal solution. The problem is that not only is CTAB cytotoxic to most cells but it also induces the sequestration of biomolecules in solution during the functionalization steps of GNRs. The presence of CTAB therefore makes it difficult to control the immobilization of biomolecules to GNRs while removing CTAB from the colloidal solution, leading to the aggregation of GNRs. The sequestration effect of ssDNA in solution by CTAB was studied in detail as a function of the CTAB concentration and the nature of the solution (water or buffer) using Forster resonance energy transfer as a detection tool. The conditions in which DNA sequestration did and did not occur could be clearly defined. Using gel electrophoresis, we could demonstrate how strongly the ssDNA sequestration effect in solution impacts the GNR surface biofunctionalization.