A　high-throughput　and　simple　chemiluminescence　(CL)　enzyme　immunoassay　(CLEIA)　for　the　determination　of　progesterone　(P)　in　human　serum　was　developed,　with　the　highly　sensitive　4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-　3,2′-adamantane)　(AMPPD)-alkaline　phosphatase　(ALP)　system　as　the　CL　detection　system.　The　results　showed　that　the　indirect　immobilization　of　rabbit　anti-progesterone　polyclonal　antibody　(RAPA)　through　secondary　antibody　exhibited　apparent　advantages　over　direct　coating　in　terms　of　antibody　saving　and　improvement　of　the　coating　stability　and　uniformity.　The　direct　analysis　of　P　in　human　serum　without　extraction　was　realized　by　using　8-anilino-1-　naphthalenesulphonic　acid　(ANS)　to　displace　P　from　its　binding　proteins.　The　effect　of　several　relevant　parameters　of　the　immunoreaction　were　examined　and　optimized.　Compared　with　some　commercial　progesterone　kits,　the　presented　CLEIA　has　higher　sensitivity　with　detection　limitation　as　low　as　0.06　ng/mL.　The　recoveries　were　95.9-101%.　The　coefficient　of　variation　was　＜8.4%　and　9.9%　for　intra-　and　inter-assay　precision,　respectively.　This　method　has　been　successfully　applied　to　the　evaluation　of　P　in　human　serum.　Copyright　©　2008　John　Wiley　&　Sons,　Ltd.