Objective To relate serum and follicular fluid (FF) kisspeptin and estradiol levels in different stages of stimulation during Intracytoplasmic Sperm Injection (ICSI) with oocyte maturity and endometrial thickness among unexplained infertile females. Methods This cross-sectional study was carried out at the Australian Concept Infertility Medical Centre from March 2017 till March 2018. Fifty unexplained infertile females, booked for ICSI, were included in the study. Serum kisspeptin and estradiol were estimated by Enzyme-Linked Immunosorbent Assay in all four stages; 1: follicular stimulation, 2: ovulation induction, 3: oocyte pickup, and 4: embryo transfer. FF was aspirated during oocyte retrieval (stage 3) for the analysis of KP and estradiol. Pregnancy outcomes were categorized as non-pregnant, preclinical abortion, and clinical pregnancy. Results The age of the study subjects was 32.04 ± 2.29 (Mean±SD) years, with mean BMI of 28.51 ± 4.15 (Mean±SD) kg/m2. Mean serum kisspeptin and estradiol levels increased in all subjects as the stimulation proceeded stages 1–3; however, the mean dropped after retrieval of the oocytes (stage 4). Out of 27 female subjects who completed the cycle, 17 remained non-pregnant, 4 had preclinical abortion, and 6 acquired clinical pregnancy. The FF kisspeptin concentration was significantly higher than serum concentrations and positively correlated with serum and FF estradiol concentrations. FF-kisspeptin correlated with serum kisspeptin in Stage 3 (r = 0.930, p<0.001), maturity of oocyte (r = 0.511, p = 0.006) and endometrial thickness (r = 0.522, p = 0.005). Kisspeptin in stage 3 was also found to correlate with endometrial thickness (r = 0.527, p = 0.005) and with estradiol (r = 0.624, p = 0.001) independently. Conclusion Increase in serum and FF-kisspeptin and estradiol levels from stages 1 to 3, resulted in an optimum endometrial thickness, probability of fertilization of oocytes and chances of clinical pregnancy in Assisted Reproductive Techniques /ICSI cycles of unexplained infertile females.