A facile and versatile procedure for the synthesis of novel functionalised 10-(1H-indol-3-yl)acenaphtho[1,2-d]pyrimidin-8-amine (compound 4) has been described. The novel intermediate enone (3) and pyrimidine derivatives (4) were characterised by using FT-IR, NMR and MS. Moreover, the photophysical properties of compound 4 and its interaction with HSA (Human Serum Albumin) have been investigated by means of photometric and fluorimetric titration. Compound 4 showed the increase in fluorescence and hypsochromic shift of its maximum upon the addition of HSA that interaction is supported by molecular docking. Overall, Compound 4 has promising affinity towards HSA which was not previously reported for any fused acenaphtenone–pyrimidine hybrid analogues.