After exploring multi-round difference feature of Serpent encryption algorithm and using the method of constructing S-boxes algebraic equations, we put forward Rectangle algebraic attack of 10-round Serpent-256. This method analyzed that 10-round Serpent-256 needed 2×2144 ×210 = 2155 pairs of selective plaintexts, 2155 times of 10-round encryption and 2155 times of 10-round decipher, as well as memory store space of 2131.8 groups to guess 10 bits of 10-round Serpent-256 encrypted keys.