The microstructures and fibrous composition of egg cases of three middle-size spiders, Parasteatoda tepidariorum (Theridiidae), Leucauge blanda (Tetragnathidae) and Hippasa holmerae (Lycosidae) were examined using SEM technique and amino acid analysis. The shapes of the egg cases of the above-mentioned three spiders are pear-shaped, flabelliform ellipsoid and ellipsoid respectively. The outer cover of the egg case of P.tepidariorum is only composed with one type of silk fiber with even diameter, whereas outer covers of other two spiders’ egg cases mainly consist of cylindrical gland silk together with a few other glands silk. The amino acid composition of cylindrical gland silk form P. tepidariorum egg case is very different from that of conserved Spidroin of cylindrical gland from other spiders, which indicates that cylindrical gland silk consists of unknown Spidroin. It needs to be identified through the further investigation of molecular biology. According to the amino acid composition and SEM, the gland origin of silk fiber with different diameter was analysized.