Overexpression of epidermal growth factor receptor (EGFR) plays a significant role in the development and metastasis of many solid tumors. Strategies based on anti-EGFR immunotoxins have shown promising results in several studies， but immunogenicity of antibody and toxin moieties is a limitation of this type of therapeutics. In the present study， a novel humanized anti-EGFR immunotoxin (huscFv-PE25) was developed by genetic fusing of a humanized anti-EGFR single-chain variable fragment (huscFv) with a modified Pseudomonas aeruginosa exotoxin A (PE25KDEL). The reactivity and toxicity of this immunotoxin with tumor cells were assessed by dot-blot， enzyme-linked immunosorbent assay， and MTT procedures. Results of enzyme-linked immunosorbent assay and dot-blot assay indicated that the immunotoxin recognizes and efficiently binds to EGFR-overexpressing tumor cells. MTT assay showed a specific growth-inhibitory effect of huscFv-PE25 on EGFR-overexpressing A431 cells， without any inhibitory effect on EGFR-negative cells. In conclusion， the results of this study indicated that huscFv-PE25 can recognize and exert an inhibitory effect on EGFR-overexpressing cancer cells， despite its smaller size and lower immunogenicity. This may provide a basis for the development of novel clinical therapeutic agents against EGFR-overexpressing tumor cells.