The purpose of this paper is to provide experimental support for the cracking and debonding of the photoresist layers in use through the study of toughness of ZEP-520 electron beam photoresist layer with different thickness and the bonding strength between the substrate. By using nano-scratch technology to test the ZEP-520 electron beam photoresist with different thicknesses and analyzing the critical load when the photoresist begins to break and completely debond, the quantitative relation between thickness of the adhesive layer and toughness of the photoresist is studied, and the bonding strength is evaluated with the bonding energy of the photoresist and silicon substrate. In addition, a high-quality orthogonal grating with a frequency of 10,000 lines/mm is successfully fabricated on a 587 nm thick photoresist layer with the electron beam exposure technology, and the geometric phase analysis method is used to quantitatively characterize the grating pitch error. The results show that the toughness, bonding force and bonding energy of the ZEP-520 photoresist layer increase with the thickness of the photoresist layer, and the bonding energy tends to a fixed value of 0.17 J/m2 when the thickness of the photoresist layer is greater than 529 nm. Finally, the measured prepared grating pitch error is within 1.3% by the geometric phase analysis method, without cracking and debonding phenomenon. It is proposed that increasing the thickness of the photoresist an appropriate during the micro/nano molding process of ZEP-520 photoresist can effectively enhance its toughness as well as adhesion to the substrate, and reduce the cracks and debonding of the photoresist during use.