This paper presents algorithms for estimating length, location, and orientation of nanowires in a fluidic workspace using images obtained by optical section microscopy. Images containing multiple nanowires are first segmented to locate general areas of interest, which are then analyzed to determine discrete nanowire parameters. We use a set of image processing techniques to extract features of nanowire image patterns, e.g., boundary of nanowire, linear edges, and the intensity profile of nanowire’s diffraction fringes. The parameters of the features are then used to estimate length, 3D position, and 3D orientation of nanowires. A scene representing the workspace is reconstructed using the estimated attributes of nanowires, and it is constantly updated upon the capture of every image frame. We believe that the work described in this paper will be useful for assembly of nanowires using optical tweezers.