A monoclonal antibody (MAb) was produced by immunization of a BALB/c mouse with a conjugated morphine C6-hemisuccinated derivative (MHS) to cationized bovine serum albumin (cBSA). The hybridoma clones were screened by indirect ELISA using MHS-BSA. The best hybridoma clone was subcloned thrice by limiting dilution. This hybridoma was found to be of IgG2b class and subclass and contained lambda light chain. The affinity of the MAb to morphine was obtained 2.8×10(9) M(-1). The titer of the cell culture supernatant was at least 1:800. The MAb was cross-reacted with codeine (100%) and apomorphine (16.5%), but not with heroin, naloxone, naltrexone, or papaverine. Morphine was conjugated to HRP using a mixed anhydride method and a direct competitive ELISA was designed using anti-morphine MAb. The assay was sensitive over the 50 ng/mL to 5 μg/mL concentration range. In conclusion, this MAb is useful for the development of immunoassays to measure morphine in urine.