Disclosed are a reagent for endotoxin-specific assay which comprises an insoluble carrier having immobilized thereon at least an endotoxin-sensitive factor derived from a limulus amebocyte; a kit for endotoxin-specific assay containing said reagent and a substrate for activated factor C or a substrate for clotting enzyme; a method for assaying endotoxin comprising applying a sample solution to said reagent to cause endotoxin in the sample to react with factor C in said reagent and determining a change of a substrate; and a process for preparing said reagent which comprises physically or chemically immobilizing at least an endotoxin-sensitive factor derived from a limulus amebocyte on an insoluble carrier. Endotoxin in a sample, even turbid or colored, can be specifically assayed with ease and rapidness without the influence of a (1.fwdarw.3)-.beta.-glucan.