Photoelectrochemical　(PEC)　detection　is　an　emerging　and　promising　analytical　tool.　However,　its　actual　application　still　faces　some　challenges　like　potential　damage　of　biomolecules　(caused　by　itself　system)　and　intrinsic　low-throughput　detection.　To　solve　the　problems,　herein　we　design　a　novel　split-type　photoelectrochemical　immunoassay　(STPIA)　for　ultrasensitive　detection　of　prostate　specific　antigen　(PSA).　Initially,　the　immunoreaction　was　performed　on　a　microplate　using　a　secondary　antibody/primer-circular　DNA-labeled　gold　nanoparticle　as　the　detection　tag.　Then,　numerously　repeated　oligonucleotide　sequences　with　many　biotin　moieties　were　in　situ　synthesized　on　the　nanogold　tag　via　RCA　reaction.　The　formed　biotin　concatamers　acted　as　a　powerful　scaffold　to　bind　with　avidin-alkaline　phosphatase　(ALP)　conjugates　and　construct　a　nanoenzyme　reactor.　By　this　means,　enzymatic　hydrolysate　(ascorbic　acid)　was　generated　to　capture　the　photogenerated　holes　in　the　CdS　quantum　dot-sensitized　TiO2　nanotube　arrays,　resulting　in　amplification　of　the　photocurrent　signal.　To　elaborate,　the　microplate-based　immunoassay　and　the　high-throughput　detection　system,　a　semiautomatic　detection　cell　(installed　with　a　three-electrode　system),　was　employed.　Under　optimal　conditions,　the　photocurrent　increased　with　the　increasing　PSA　concentration　in　a　dynamic　working　range　from　0.001　to　3　ng　mL-1,　with　a　low　detection　limit　(LOD)　of　0.32　pg　mL-1.　Meanwhile,　the　developed　split-type　photoelectrochemical　immunoassay　exhibited　high　specificity　and　acceptable　accuracy　for　analysis　of　human　serum　specimens　in　comparison　with　referenced　electrochemiluminescence　immunoassay　method.　Importantly,　the　system　was　not　only　suitable　for　the　sandwich-type　immunoassay　mode,　but　also　utilized　for　the　detection　of　small　molecules　(e.g.,　aflatoxin　B1)　with　a　competitive-type　assay　format.　©　2015　American　Chemical　Society.