Background and Objectives Ovarian cancer remains the deadliest malignancy of the female reproductive system. The ability to identify and destroy all ovarian tumor nodules may have a termendous impact on preventing tumor recurrence, and patient survival. The objective of this study is to investigate the effectiveness of a nano-structured system for combined near infrared (NIR) fluorescence imaging of human epidermal growth factor receptor-2 (HER2) over-expression, as a biomarker of ovarian cancer cells, and photothermal destruction of these cells in vitro. Materials and Methods The nano-structured system consists of the near infrared dye, indocyanine green (ICG), encapsulated within poly(allylamine) hydrochloride chains cross-linked ionically with sodium phosphate. The surface of the construct is functionalized by covalently attached polyethylene glycol, and monoclonal antibodies against HER2 using reducitve amination methods. We use dynamic light scattering, and absorption and fluorescence spectroscopy for phyiscal characterization of the constructs. Flow cytometry and fluorescence microscopy are used to investigate molecular targeting and imaging capabilities of the constructs against SKOV3 and OVCAR3 ovarian cancer cell lines, which have relatively high and low expression levels of the HER2 receptor, respectively. Continuous NIR laser irradiation at 808 nm is used to investigating the utility of the constructs in mediating photothermal destruction of SKOV3 cells. Results Flow cytometry results indicate that the functionalized nano-constructs are more effective in targeting the HER2 receptor than non-encapsulated ICG and non-functionlaized constructs (P < 0.005). Fluorescence microscopic images show the capaiblity of the functionalized constructs in NIR imaging of HER2 overexpression. The functionalized nano-constructs are also capable of inducing a significantly greater increase in photothermal destruction of SKOV3 cells than free ICG and non-functionalized constructs (P < 0.005). Conclusion We have demonstrated the efficacy of polymeric nano-structured constructs loaded with ICG, and functionalized with the monoclonal antibodies, as thernaostic materials for targted molecular NIR imaging of the HER2 receptor overexpression on ovarian cancer cells, and photothermal destruction of these cells. These nanoparticles may prove useful towards intraoperative detection, imaging, and phototherapy of small ovarian cancer nodules. © 2014 Wiley Periodicals, Inc.