Apart from its vital function as a redox cofactor, nicotinamide adenine dinucleotide (NAD⁺ ) has emerged as a crucial substrate for NAD⁺ -consuming enzymes, including poly(ADP-ribosyl)transferase 1 (PARP1) and CD38/CD157. Their association with severe diseases, such as cancer, Alzheimer's disease, and depressions, necessitates the development of new analytical tools based on traceable NAD⁺ surrogates. Here, the synthesis, photophysics and biochemical utilization of an emissive, thieno[3,4-d]pyrimidine-based NAD⁺ surrogate, termed N^th AD⁺ , are described. Its preparation was accomplished by enzymatic conversion of synthetic ^th ATP by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1). The new NAD⁺ analogue possesses useful photophysical features including redshifted absorption and emission maxima as well as a relatively high quantum yield. Serving as a versatile substrate, N^th AD⁺ was reduced by alcohol dehydrogenase (ADH) to N^th ADH and afforded ^th ADP-ribose (^th ADPr) upon hydrolysis by NAD⁺ -nucleosidase (NADase). Furthermore, N^th AD⁺ was engaged in cholera toxin A (CTA)-catalyzed mono(^th ADP-ribosyl)ation, but was found incapable in promoting PARP1-mediated poly(^th ADP-ribosyl)ation. Due to its high photophysical responsiveness, N^th AD⁺ is suited for spectroscopic real-time monitoring. Intriguingly, and as an N7-lacking NAD⁺ surrogate, the thieno-based cofactor showed reduced compatibility (i.e., functional similarity compared to native NAD⁺ ) relative to its isothiazolo-based analogue. The distinct tolerance, displayed by diverse NAD⁺ producing and consuming enzymes, suggests unique biological recognition features and dependency on the purine N7 moiety, which is found to be of importance, if not essential, for PARP1-mediated reactions.