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In this paper, the local stiffness of alive Caenorhabditis elegans (C. elegans) is evaluated by environmental-scanning electron microscope (E-SEM) nanorobotic manipulation system. The local and surface stiffness of C. elegans is important to evaluate to inject or insert micro-nano probes for gene-deliver or electrophysiologic applications. At first, we observe their fine structures by E-SEM system directly; without any drying or dyeing processes. The viability of C. elegans inside E-SEM is determined from the motion of C. elegans before and after setting inside the E-SEM. The local stiffness of C. elegans is evaluated from the deformation area by applying the forces with nanoprobes. The nanoprobes were fabricated by focus ion beam (FIB) etching at the tip of atomic force microscope (AFM) cantilever. The measurement position can arbitrarily be controlled by the nanomanipulator under E-SEM observation. The local stiffness distribution of C. elegans was evaluated on its head, body (on/around alea) and tail. From the experimental results, the measured rigidity on lateral alae was higher than it around lateral alae. This local stiffness measurement information is important for nano-surgery, gene-deliver or electrophysiologic applications and novel cell diagnosis.