It is postulated that the outcomes in treating breast cancer with intraoperative radiotherapy (IORT) would be affected by the residual cancer cell distribution within the tumor bed. The three‐dimensional (3D) radiation doses of IntrabeamTM (IB) IORT with a 4‐cm spherical applicator at the energy of 50 and 40 kV were calculated. The modified linear quadratic model (MLQ) was used to estimate the radiobiological responses of the cancer cells and interspersed normal tissues with various radiosensitivities. By comparing the average survival fraction of normal tissues in IB‐IORT and uniform dose treatment for the same level of cancer cell killing, the therapeutic ratios (TRs) were derived. The equivalent uniform dose (EUD) was found to increase with the prescription dose and decrease with the cancer cell infiltrating distance. For 50 kV beam at the 20 Gy prescription dose, the EUDs are 18.03, 16.49 and 13.56, 11. 29, and 9.28 Gy respectively, for 1.5, 3.0, 6.0, 9, and 15.0 mm of the cancer cell infiltrating distance into surrounding tissue. The dose rate of 50 kV is at least 1.87× higher than that of 40 kV beam. The EUDs of 50 kV beam are up to 15% higher than that of the 40 kV beam. The TR increases with the prescription dose, but decreases with the distance of cancer cell infiltration distance. Average TRs of 50 kV beam are up to 30% larger than that of 40 kV beam. In conclusion, IB‐IORT can provide a possible therapeutic advantage on sparing more normal tissue compared with the External Beam IORT (EB‐IORT) for shallowly populated unicentric breast lesion. Our data suggest that IB‐IORT dose size should be adjusted based on the individual patient's cancer cell infiltrating distance for delivering an effective dose, one dose‐fits‐all regimen may have undertreated some patients with large cancer infiltrating distance.