Tensile tests of single red blood cells have been conducted to determine their fracture strain for the development of a hemolysis simulator. The red blood cells were however detached from holding tools before their membrane broke down due to their high elasticity. The present study therefore examines means and conditions to firmly hold single red blood cells in the tensile tests. We tested urethane and epoxy resin as a glue, and examined a needle and a micropipette as a holding tool. Tensile tests were repeated with various combinations of the glue and the holding tool. Examined parameters for the glues were a dry time in the air (Ta), an underwater hardening time (Tw) and a fixation time (Tf). The fracture of single red blood cells was achieved when the urethane was used as a glue and the micropipette was adopted as the holding tool. The red blood cell failed via fracture at a deformation ratio of 5.32. In this achievement, Ta, Tw and Tf were 300 s, 90 s and 1800 s, respectively. The results suggested use of urethane as a glue and micropipette as the holding tool for determining the fracture strain of the red blood cells by tensile tests.