My application which is hosted on EC2 instance which is in private subnet is not connecting with RDS which is in private subnet

Hello.

If you encounter an error when connecting, could you please share the error message?
Also, could you please share your EC2 security group settings and RDS security group settings?

If you get a timeout error, you may need to check the settings of your security group, subnet network ACL, Windows firewall, etc.
https://repost.aws/knowledge-center/rds-cannot-connect

You also need to make sure that "enableDnsHostnames" and "enableDnsSupport" are enabled in your VPC.
https://docs.aws.amazon.com/vpc/latest/userguide/vpc-dns.html#vpc-dns-support
https://docs.aws.amazon.com/AmazonRDS/latest/UserGuide/USER_VPC.WorkingWithRDSInstanceinaVPC.html

If you want your DB instance in the VPC to be publicly accessible, make sure to turn on the VPC attributes DNS hostnames and DNS resolution.

Ec2 instance(windows) and RDS MySQL both are in same vpc same region and on private subnet

Can you confirm that the EC2 instance and RDS instance are in the same subnet?

Tried by adding security group but it doesnt help.

Which security group settings did you try? Assuming that MySQL is running on its default port 3306, your EC2 instance will need a security group with an outbound rule allowing TCP port 3306 to the RDS instance's private IP address (or subnet, or just make it 0.0.0.0/0 for troubelshooting, you can tighten it later). On the RDS instance, it will need a security group associated with it which has an inbound rule allowing TCP port 3306 from the EC2 instance's private IP address (or subnet, or again just make it 0.0.0.0/0 to get it working).